F HeLa cells stably expressing GFP - Nup107 were synchronized by double thymidine block and released for 12 hours , permeabilized with Triton / SDS or digitonin for antibodies to access the nuclear and cytoplasmic or cytoplamsic side of the nucleus , respectively , and analysed by immunofluorescence microscopy .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F HeLa cells stably expressing GFP-Nup107 were synchronized by double thymidine block and released for 12 hours, permeabilized with Triton/SDS or digitonin for antibodies to access the nuclear and cytoplasmic or cytoplamsic side of the nucleus, respectively, and analysed by immunofluorescence microscopy.


A HeLa cells were treated with the indicated siRNAs , synchronized by double thymidine block and release for 12 hours and analysed by immunofluorescence microscopy . The magnified framed regions are shown in the corresponding numbered panels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block and release for 12 hours and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels.


B HeLa cells stably expressing GFP , GFP - FXR1 wild type ( WT ) and GFP - FXR1 mutated in the sequence recognized by FXR1 siRNA - 1 ( GFP - FXR1 - MUT - siRNA1 ) were treated with the indicated siRNAs , synchronized by double thymidine block , released for 24 hours and then analysed by immunofluorescence microscopy . The percentage of cells with cytoplasmic nucleoporin granules was quantified , 1000 cells were analysed for each graph ( mean ± SD , * * P < 0 . 01 ; * * * P < 0 . 001 , N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B HeLa cells stably expressing GFP, GFP-FXR1 wild type (WT) and GFP-FXR1 mutated in the sequence recognized by FXR1 siRNA-1 (GFP-FXR1-MUT-siRNA1) were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 hours and then analysed by immunofluorescence microscopy. The percentage of cells with cytoplasmic nucleoporin granules was quantified, 1000 cells were analysed for each graph (mean ±SD, **P < 0.01; ***P < 0.001, N = 3).


HeLa cells were treated with the indicated siRNAs , synchronized by double thymidine block , released for 12 hours and analysed by immunofluorescence microscopy . Nups present in different NPC subcomplexes are depicted in the color code corresponding to the NPC scheme shown on the right . Additional or complementary representative images and channels of cells depicted in ( C )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Nups present in different NPC subcomplexes are depicted in the color code corresponding to the NPC scheme shown on the right. Additional or complementary representative images and channels of cells depicted in (C)


HeLa cells were treated with the indicated siRNAs , synchronized by double thymidine block , released for 12 hours and analysed by immunofluorescence microscopy . Nuclear intensity of FG - Nups labelled by mAb414 ( D ) , RanBP2 ( E ) and GFP - Nup107 ( F ) was quantified . 1800 cells were analysed for each graph ( mean ± SD , * P < 0 . 05 ; * * P < 0 . 01 ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Nuclear intensity of FG-Nups labelled by mAb414 (D), RanBP2 (E) and GFP-Nup107 (F) was quantified. 1800 cells were analysed for each graph (mean ±SD, *P < 0.05; **P < 0.01; N = 3).


G , H Asynchronously proliferating U2OS cells were treated with the indicated siRNAs and analysed by immunofluorescence microscopy . The percentage of cells with cytoplasmic nucleoporin granules ( G ) was quantified and nuclear intensity of FG - Nups labelled by mAb414 ( H ) was quantified . 1600 cells were analysed in ( G ) and 2100 cells were analysed in ( H ) ( mean ± SD , * P < 0 . 05 ; * * * P < 0 . 001 ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G, H Asynchronously proliferating U2OS cells were treated with the indicated siRNAs and analysed by immunofluorescence microscopy. The percentage of cells with cytoplasmic nucleoporin granules (G) was quantified and nuclear intensity of FG-Nups labelled by mAb414 (H) was quantified. 1600 cells were analysed in (G) and 2100 cells were analysed in (H) (mean ±SD, *P < 0.05; *** P<0.001; N = 3).


A HeLa cells were treated with the indicated siRNAs , synchronized by double thymidine block , released for 24 hours and analysed by immunofluorescence microscopy . The magnified framed regions are shown in the corresponding numbered panels . Arrowheads point to nuclear blebs observed in FXR1 - deficient cells .

A HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 hours and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels. Arrowheads point to nuclear blebs observed in FXR1-deficient cells.


B , HeLa cells stably expressing GFP , GFP - FXR1 wild type ( WT ) and GFP - FXR1 mutated in the sequence recognized by FXR1 siRNA - 1 ( GFP - FXR1 - MUT - siRNA1 ) were treated with the indicated siRNAs , synchronized by double thymidine block , released for 24 hours and analysed by Western blot ( B )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B, HeLa cells stably expressing GFP, GFP-FXR1 wild type (WT) and GFP-FXR1 mutated in the sequence recognized by FXR1 siRNA-1 (GFP-FXR1-MUT-siRNA1) were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 hours and analysed by Western blot (B)


C HeLa cells stably expressing GFP , GFP - FXR1 wild type ( WT ) and GFP - FXR1 mutated in the sequence recognized by FXR1 siRNA - 1 ( GFP - FXR1 - MUT - siRNA1 ) were treated with the indicated siRNAs , synchronized by double thymidine block , released for 24 hours and analysed by immunofluorescence microscopy ( C ) . The percentage of cells with irregular nuclei was quantified , 1000 cells were analysed ( mean ± SD , * * P < 0 . 01 , * * * P < 0 . 001 ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C HeLa cells stably expressing GFP, GFP-FXR1 wild type (WT) and GFP-FXR1 mutated in the sequence recognized by FXR1 siRNA-1 (GFP-FXR1-MUT-siRNA1) were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 hours and analysed by immunofluorescence microscopy (C). The percentage of cells with irregular nuclei was quantified, 1000 cells were analysed (mean ±SD, **P < 0.01, ***P < 0.001; N = 3).


HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs , synchronized by double thymidine block , released for 12 hours and analysed by immunofluorescence microscopy . Time from prophase till anaphase ( D ) , from prophase till metaphase ( E ) , from metaphase till anaphase ( F ) and from anaphase till chromatin decondensation ( G ) was quantified .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Time from prophase till anaphase (D), from prophase till metaphase (E), from metaphase till anaphase (F) and from anaphase till chromatin decondensation (G) was quantified.


HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs , synchronized by double thymidine block , released for 12 hours and analysed by immunofluorescence microscopy . The selected frames of the movies are depicted and time is shown in minutes ( H ) . Arrowheads point to nuclear blebs appearing during nuclear expansion of FXR1 - deficient cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. The selected frames of the movies are depicted and time is shown in minutes (H). Arrowheads point to nuclear blebs appearing during nuclear expansion of FXR1-deficient cells.


HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs , synchronized by double thymidine block , released for 12 hours and analysed by immunofluorescence microscopy . Percentage of daughter cells with irregular nuclei was quantified in ( I )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Percentage of daughter cells with irregular nuclei was quantified in (I)


HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs , synchronized by double thymidine block , released for 12 hours and analysed by immunofluorescence microscopy . time from anaphase till nuclear blebs was quantified in ( J ) .

HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. time from anaphase till nuclear blebs was quantified in (J).


A - D HeLa cells stably expressing GFP - Nup107 were treated with indicated siRNAs , synchronized by double thymidine block , released and analysed by live video spinning disk confocal microscopy ( A ) . The selected frames of the movies are depicted and time is shown in minutes . The onset of anaphase is indicated . The magnified framed regions with time indicated in minutes are shown in ( B ) . White arrowheads point to the cytoplasmic GFP - NUP107 granules appearing during nuclear expansion of control and FXR1 - deficient cells , yellow arrowheads point to the fussion events of GFP - NUP107 granules with NE in control cells . The percentage of cells with cytoplasmic GFP - Nup107 granules was quantified in ( C ) . Time from anaphase till GFP - Nup107 cytoplasmic granule formation was quantified in ( D ) . 57 cells were analysed ( mean ± SD , * P < 0 . 05 ; ns = non - significant ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-D HeLa cells stably expressing GFP-Nup107 were treated with indicated siRNAs, synchronized by double thymidine block, released and analysed by live video spinning disk confocal microscopy (A). The selected frames of the movies are depicted and time is shown in minutes. The onset of anaphase is indicated. The magnified framed regions with time indicated in minutes are shown in (B). White arrowheads point to the cytoplasmic GFP-NUP107 granules appearing during nuclear expansion of control and FXR1-deficient cells, yellow arrowheads point to the fussion events of GFP-NUP107 granules with NE in control cells. The percentage of cells with cytoplasmic GFP-Nup107 granules was quantified in (C). Time from anaphase till GFP-Nup107 cytoplasmic granule formation was quantified in (D). 57 cells were analysed (mean ±SD, *P < 0.05; ns = non-significant; N = 3).


A HeLa cells stably expressing GFP - Nup133 were synchronized by double thymidine block , released for 12 hours , treated or not with NaAsO2 to induce stress granule formation and with 1 , 6 Hexanediol , and analysed by immunofluorescence microscopy . The magnified framed regions are shown in the corresponding numbered panels .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

A HeLa cells stably expressing GFP-Nup133 were synchronized by double thymidine block, released for 12 hours, treated or not with NaAsO2 to induce stress granule formation and with 1,6 Hexanediol, and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels.


B , C HeLa cells stably expressing GFP - Nup133 were treated with the indicated siRNAs , synchronized by double thymidine block , released for 12 hours , treated with or without 1 , 6 Hexanediol and analysed by immunofluorescence microscopy . The magnified framed regions are shown in the corresponding numbered panels . The percentage of cells with cytoplasmic GFP - Nup133 granules was quantified in ( C ) , 3100 cells were analysed ( mean ± SD , * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B, C HeLa cells stably expressing GFP-Nup133 were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours, treated with or without 1,6 Hexanediol and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels. The percentage of cells with cytoplasmic GFP-Nup133 granules was quantified in (C), 3100 cells were analysed (mean ±SD, **P < 0.01; ***P < 0.001; ****P < 0.0001; N = 3).


A HeLa cells stably expressing GFP alone or GFP - FXR1 were immunoprecipitated using GFP - Trap beads ( GFP - IP ) , analysed by Western blot and quantified ( mean , * P < 0 . 05 ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A HeLa cells stably expressing GFP alone or GFP-FXR1 were immunoprecipitated using GFP-Trap beads (GFP-IP), analysed by Western blot and quantified (mean, *P < 0.05; N = 3).


B , C HeLa cells were treated with the indicated siRNAs , synchronized by double thymidine block , released for 12 hours and analysed by immunofluorescence microscopy . Arrowheads indicate blebbed regions of nuclei . The percentage of interphasic cells with Nup granules was quantified ( C ) , 900 cells were analysed ( mean ± SD , * * * P < 0 . 001 ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B, C HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Arrowheads indicate blebbed regions of nuclei. The percentage of interphasic cells with Nup granules was quantified (C), 900 cells were analysed (mean ±SD, ***P < 0.001; N = 3).


D Lysates of HeLa cells stably expressing GFP alone or GFP - FXR1 were subjected to immunoprecipitation using GFP - Trap beads ( GFP - IP ) , analysed by Western blot and quantified ( mean , * P < 0 . 05 ; * * P < 0 . 01 ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Lysates of HeLa cells stably expressing GFP alone or GFP-FXR1 were subjected to immunoprecipitation using GFP-Trap beads (GFP-IP), analysed by Western blot and quantified (mean, *P < 0.05; **P < 0.01; N = 3).


E , F HeLa cells were treated with the indicated siRNAs , synchronized by double thymidine block and released for 12 hours and analysed by immunofluorescence microscopy for FXR1 and mAb414 . The magnified framed regions are shown in the corresponding numbered panels in ( E ) . The percentage of cells with cytoplasmic Nup granules was quantified in ( F ) , 2700 cells were analysed ( mean ± SD , * * * P < 0 . 001 ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, F HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block and released for 12 hours and analysed by immunofluorescence microscopy for FXR1 and mAb414. The magnified framed regions are shown in the corresponding numbered panels in (E). The percentage of cells with cytoplasmic Nup granules was quantified in (F), 2700 cells were analysed (mean ±SD, ***P < 0.001; N = 3).


A , B HeLa cells stably expressing GFP - Nup133 were treated with the indicated siRNAs , synchronized by double thymidine block , released for 12 hours , treated with nocodazole to induce granule formation and washed - out as indicated and analysed by immunofluorescence microscopy . The percentage of cells with cytoplasmic GFP - Nup133 granules was quantified in ( B ) , 5200 cells were analysed ( mean ± SD , * * P < 0 . 01 ; * * * P < 0 . 001 ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B HeLa cells stably expressing GFP-Nup133 were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours, treated with nocodazole to induce granule formation and washed-out as indicated and analysed by immunofluorescence microscopy. The percentage of cells with cytoplasmic GFP-Nup133 granules was quantified in (B), 5200 cells were analysed (mean ±SD, **P < 0.01; ***P < 0.001; N = 3).


C - E HeLa cells stably expressing GFP - Nup107 were treated with the indicated siRNAs , synchronized by double thymidine block , released for 12 hours , treated with nocodazole to induce granule formation and washed - out as in ( A ) and analysed by live video spinning disk confocal microscopy . The selected frames of the movies are depicted and time is shown in minutes . The magnified framed regions are depicted in the lower rows . White arrowheads point to individual GFP - Nup107 - positive granules . Yellow arrowheads point to the granules undergoing fusion events . The percentage of cells with fusion / fission events of GFP - Nup107 granules was quantified in ( D ) , 815 cells were analysed ( mean ± SD , * * P < 0 . 01 ; N = 3 ) . The number of fusion / fission events per cell was quantified in ( E ) , 815 cells were analysed ( mean ± SD ; N = 3 ) .

C-E HeLa cells stably expressing GFP-Nup107 were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours, treated with nocodazole to induce granule formation and washed-out as in (A) and analysed by live video spinning disk confocal microscopy. The selected frames of the movies are depicted and time is shown in minutes. The magnified framed regions are depicted in the lower rows. White arrowheads point to individual GFP-Nup107-positive granules. Yellow arrowheads point to the granules undergoing fusion events. The percentage of cells with fusion/fission events of GFP-Nup107 granules was quantified in (D), 815 cells were analysed (mean ±SD, **P < 0.01; N = 3). The number of fusion/fission events per cell was quantified in (E), 815 cells were analysed (mean ±SD; N = 3).


Human FXS - patient derived fibroblasts ( FXS - 1 , FXS - 2 ) and control human fibroblasts were synchronized in early G1 by Monastrol release and analysed by Western blot ( A )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Human FXS-patient derived fibroblasts (FXS-1, FXS-2) and control human fibroblasts were synchronized in early G1 by Monastrol release and analysed by Western blot (A)


Human FXS - patient derived fibroblasts ( FXS - 1 , FXS - 2 ) and control human fibroblasts were synchronized in early G1 by Monastrol release and analysed by immunofluorescence microscopy Examples of Nup localization defects are shown in ( B )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Human FXS-patient derived fibroblasts (FXS-1, FXS-2) and control human fibroblasts were synchronized in early G1 by Monastrol release and analysed by immunofluorescence microscopy Examples of Nup localization defects are shown in (B)


Human FXS - patient derived fibroblasts ( FXS - 1 , FXS - 2 ) and control human fibroblasts were synchronized in early G1 by Monastrol release and analysed by immunofluorescence microscopy The percentage of cells with cytoplasmic Nup granules was quantified in ( C ) , 283 cells were analysed ( mean ± SD , * * * P < 0 . 001 ; N = 3 ) .

Human FXS-patient derived fibroblasts (FXS-1, FXS-2) and control human fibroblasts were synchronized in early G1 by Monastrol release and analysed by immunofluorescence microscopy The percentage of cells with cytoplasmic Nup granules was quantified in (C), 283 cells were analysed (mean ±SD, ***P < 0.001; N = 3).


Human FXS - patient derived fibroblasts ( FXS - 1 , FXS - 2 ) and control human fibroblasts were synchronized in early G1 by Monastrol release and analysed by immunofluorescence microscopy examples of nuclear architecture defects are shown in ( D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Human FXS-patient derived fibroblasts (FXS-1, FXS-2) and control human fibroblasts were synchronized in early G1 by Monastrol release and analysed by immunofluorescence microscopy examples of nuclear architecture defects are shown in (D).


Human induced pluripotent stem cells ( iPSCs ) derived from a FXS patient ( FXS - iPSC ) and the isogenic rescue cells ( C1 _ 2 - iPSC ) were analysed by immunofluorescence

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Human induced pluripotent stem cells (iPSCs) derived from a FXS patient (FXS-iPSC) and the isogenic rescue cells (C1_2-iPSC) were analysed by immunofluorescence microscopy


Human induced pluripotent stem cells ( iPSCs ) derived from a FXS patient ( FXS - iPSC ) and the isogenic rescue cells ( C1 _ 2 - iPSC ) were analysed by immunofluorescence microscopy The percentage of cells with cytoplasmic Nup133 granules was quantified in ( F ) , 5500 cells were analysed ( mean ± SD , * * P < 0 . 01 ; N = 3 ) .

Human induced pluripotent stem cells (iPSCs) derived from a FXS patient (FXS-iPSC) and the isogenic rescue cells (C1_2-iPSC) were analysed by immunofluorescence microscopy The percentage of cells with cytoplasmic Nup133 granules was quantified in (F), 5500 cells were analysed (mean ±SD, **P < 0.01; N = 3).


Human induced pluripotent stem cells ( iPSCs ) derived from a FXS patient ( FXS - iPSC ) and the isogenic rescue cells ( C1 _ 2 - iPSC ) were analysed by immunofluorescence microscopy Examples of co - localization events of re - expressed FMRP and Nup133 are shown in ( G ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Human induced pluripotent stem cells (iPSCs) derived from a FXS patient (FXS-iPSC) and the isogenic rescue cells (C1_2-iPSC) were analysed by immunofluorescence microscopy Examples of co-localization events of re-expressed FMRP and Nup133 are shown in (G).


H , I Mouse Embryonic Fibroblasts ( MEFs ) derived from the Fmr1 knock - out ( KO ) mice and wild type controls were synchronized in early G1 by Monastrol release and analysed by immunofluorescence microscopy ( H ) . The percentage of cells with cytoplasmic nucleoporin granules was quantified in ( I ) , 2400 cells were analysed ( mean ± SD , * P < 0 . 05 ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H, I Mouse Embryonic Fibroblasts (MEFs) derived from the Fmr1 knock-out (KO) mice and wild type controls were synchronized in early G1 by Monastrol release and analysed by immunofluorescence microscopy (H). The percentage of cells with cytoplasmic nucleoporin granules was quantified in (I), 2400 cells were analysed (mean ±SD, *P < 0.05; N = 3).


A HeLa cells were transfected with the import / export reporter plasmid XRGG - GFP , treated with the indicated siRNAs and synchronized in early G1 phase by Monastrol release . Dexamethasone was added for 3 hours to induce XRGG - GFP nuclear import . Following wash - out the nuclear export of XRGG - GFP was analysed by live video spinning disk confocal microscopy . The selected frames of the movies are depicted in Figure EV5C . The percentage of cytoplasmic XRGG - GFP over time was quantified in Figure EV5D and quantifications of individual cells from the 20 and 30 minutes time points are depicted in ( A ) , 199 cells were analysed ( mean ± SD , * P < 0 . 05 ; * * P < 0 . 01 ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A HeLa cells were transfected with the import/export reporter plasmid XRGG-GFP, treated with the indicated siRNAs and synchronized in early G1 phase by Monastrol release. Dexamethasone was added for 3 hours to induce XRGG-GFP nuclear import. Following wash-out the nuclear export of XRGG-GFP was analysed by live video spinning disk confocal microscopy. The selected frames of the movies are depicted in Figure EV5C. The percentage of cytoplasmic XRGG-GFP over time was quantified in Figure EV5D and quantifications of individual cells from the 20 and 30 minutes time points are depicted in (A), 199 cells were analysed (mean ±SD, *P < 0.05; **P < 0.01; N = 3).


B HeLa cells stably expressing GFP - Nup133 were treated with the indicated siRNAs , synchronized by double thymidine block , released for 12 hours and analysed by immunofluorescence microscopy . The magnified framed regions are shown in the corresponding numbered panels .

B HeLa cells stably expressing GFP-Nup133 were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels.


C , D Asynchronously proliferating HeLa cells were treated with indicated siRNAs and analysed by immunofluorescence microscopy ( C ) . The percentage of p - Rb positive cells was quantified in ( D ) , 2800 cells were analysed ( mean ± SD , * * P < 0 . 01 ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D Asynchronously proliferating HeLa cells were treated with indicated siRNAs and analysed by immunofluorescence microscopy (C). The percentage of p-Rb positive cells was quantified in (D), 2800 cells were analysed (mean ±SD, **P < 0.01; N = 3).


Asynchronously proliferating HeLa cells were treated with indicated siRNAs , incubated with EdU during 30min and analysed by immunofluorescence microscopy . The percentage of p - Rb and / or EdU positive cells was quantified in ( E ) , 2100 cells were analysed ( mean ± SD , * P < 0 . 05 ; * * P < 0 . 01 ; N = 3 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Asynchronously proliferating HeLa cells were treated with indicated siRNAs, incubated with EdU during 30min and analysed by immunofluorescence microscopy. The percentage of p-Rb and/or EdU positive cells was quantified in (E), 2100 cells were analysed (mean ±SD, *P < 0.05; **P < 0.01; N = 3)


F Asynchronously proliferating HeLa cells were treated with indicated siRNAs , incubated with EdU during 30min and analysed by immunofluorescence microscopy . the percentage of p - Rb and / or cyclin B positive cells was quantified in (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Asynchronously proliferating HeLa cells were treated with indicated siRNAs, incubated with EdU during 30min and analysed by immunofluorescence microscopy. the percentage of p-Rb and/or cyclin B positive cells was quantified in (


Activated GFAP + astrocytes in white matter MS lesions with inflammatory infiltrates and demyelination show an upregulation of OTUB1 ( arrows ) . Photomicrographs are obtained from case 9 ( A ) and case 5 ( B ) listed in Table EV1 . Data are representative for all 10 MS patients analyzed . Double immunofluorescence with rabbit anti - OTUB1 ( Cy3 ) and mouse anti - GFAP ( FITC ) ; original magnification × 400 ; scale bars correspond to 50 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Activated GFAP+ astrocytes in white matter MS lesions with inflammatory infiltrates and demyelination show an upregulation of OTUB1 (arrows). Photomicrographs are obtained from case 9 (A) and case 5 (B) listed in Table EV1. Data are representative for all 10 MS patients analyzed. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-GFAP (FITC); original magnification ×400; scale bars correspond to 50 µm.


WB analysis of OTUB1 expression in cultured primary astrocytes from OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice .

O O O O O O O O O O O O O O O O O O O O O O

WB analysis of OTUB1 expression in cultured primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice.


Direct ex vivo WB analysis of OTUB1 expression in astrocytes isolated from adult OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice .

O O O O O O O O O O O O O O O O O O O O O O O O O

Direct ex vivo WB analysis of OTUB1 expression in astrocytes isolated from adult OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice.


Normal CNS architecture in OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice . Myelination is normal in the brain and spinal cord of an OTUB1fl / fl and a GFAP - Cre OTUB1fl / fl mouse . CV - LFB staining ; original magnification × 50 ; scale bars correspond to 100 µm . All photographs are representative of three mice per group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Normal CNS architecture in OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice. Myelination is normal in the brain and spinal cord of an OTUB1fl/fl and a GFAP-Cre OTUB1fl/fl mouse. CV-LFB staining; original magnification ×50; scale bars correspond to 100 µm. All photographs are representative of three mice per group.


Absolute numbers of CD45 + cells in the spinal cord of OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice ( n = 5 for both groups , mean + SEM ) . Absolute numbers of different subpopulations of leukocytes infiltrating the spinal cord of OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice were analyzed by flow cytometry ( n = 5 for both groups , mean + SEM ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Absolute numbers of CD45+ cells in the spinal cord of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice (n = 5 for both groups, mean + SEM). Absolute numbers of different subpopulations of leukocytes infiltrating the spinal cord of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were analyzed by flow cytometry (n = 5 for both groups, mean + SEM).


In an OTUB1fl / fl and a GFAP - Cre OTUB1fl / fl mouse , OTUB1 is strongly expressed by NeuN + neurons in the brain and spinal cord . Double immunofluorescence with rabbit anti - OTUB1 ( Cy3 ) and mouse anti - NeuN ( FITC ) . Single NogoA + oligodendrocytes in the brain and spinal cord ( arrows ) of an OTUB1fl / fl and a GFAP - Cre OTUB1fl / fl mouse express OTUB1 . Double immunofluorescence with rabbit anti - OTUB1 ( Cy3 ) and mouse anti - NogoA ( Alexa Fluor 488 ) . GFAP + astrocytes are of normal morphology and distribution in the brain and spinal cord of a non - immunized OTUB1fl / fl mouse and a GFAP - Cre OTUB1fl / fl mouse . Note that GFAP + astrocytes of an OTUB1fl / fl mouse do not express OTUB1 . GFAP + astrocytes of a GFAP - Cre OTUB1fl / fl mouse are also OTUB1 - negative and represent a negative control for OTUB1 staining . Double immunofluorescence with rabbit anti - OTUB1 ( Cy3 ) and mouse anti - GFAP ( FITC ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

In an OTUB1fl/fl and a GFAP-Cre OTUB1fl/fl mouse, OTUB1 is strongly expressed by NeuN+ neurons in the brain and spinal cord. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-NeuN (FITC). Single NogoA+ oligodendrocytes in the brain and spinal cord (arrows) of an OTUB1fl/fl and a GFAP-Cre OTUB1fl/fl mouse express OTUB1. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-NogoA (Alexa Fluor 488). GFAP+ astrocytes are of normal morphology and distribution in the brain and spinal cord of a non-immunized OTUB1fl/fl mouse and a GFAP-Cre OTUB1fl/fl mouse. Note that GFAP+ astrocytes of an OTUB1fl/fl mouse do not express OTUB1. GFAP+ astrocytes of a GFAP-Cre OTUB1fl/fl mouse are also OTUB1-negative and represent a negative control for OTUB1 staining. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-GFAP (FITC).


Activated GFAP + astrocytes in the spinal cord upregulate OTUB1 in EAE at maximal disease activity ( day 15 p . i . ) in an OTUB1fl / fl mouse ( arrowheads ) , but not in a GFAP - Cre OTUB1fl / fl mouse . Note the OTUB1 - expressing neuron in an OTUB1fl / fl mouse which is surrounded by GFAP - expressing processes of an activated astrocyte ( asterisk ) . At day 22 p . i . , astrocytes have downregulated OTUB1 expression while neurons express OTUB1 in an OTUB1fl / fl mouse ( asterisk ) . Astrocytes in a GFAP - Cre OTUB1fl / fl mouse are OTUB1 - negative while neurons express OTUB1 ( asterisk ) . At day 22 p . i . , demyelination in a GFAP - Cre OTUB1fl / fl mouse is much more severe and extended as compared to an OTUB1fl / fl mouse . Inflammation persists in the GFAP - Cre OTUB1fl / fl mouse , whereas it has resolved in an OTUB1fl / fl mouse . All photographs are representative of three mice per group ; original magnification × 400 ; scale bars correspond to 50 µm . ( A , C ) Double immunofluorescence with rabbit anti - OTUB1 ( Cy3 ) and mouse anti - GFAP ( FITC ) . ( B , D ) CV - LFB staining .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Activated GFAP+ astrocytes in the spinal cord upregulate OTUB1 in EAE at maximal disease activity (day 15 p.i.) in an OTUB1fl/fl mouse (arrowheads), but not in a GFAP-Cre OTUB1fl/fl mouse. Note the OTUB1-expressing neuron in an OTUB1fl/fl mouse which is surrounded by GFAP-expressing processes of an activated astrocyte (asterisk). At day 22 p.i., astrocytes have downregulated OTUB1 expression while neurons express OTUB1 in an OTUB1fl/fl mouse (asterisk). Astrocytes in a GFAP-Cre OTUB1fl/fl mouse are OTUB1-negative while neurons express OTUB1 (asterisk). At day 22 p.i., demyelination in a GFAP-Cre OTUB1fl/fl mouse is much more severe and extended as compared to an OTUB1fl/fl mouse. Inflammation persists in the GFAP-Cre OTUB1fl/fl mouse, whereas it has resolved in an OTUB1fl/fl mouse. All photographs are representative of three mice per group; original magnification ×400; scale bars correspond to 50 µm. (A, C) Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-GFAP (FITC). (B, D) CV-LFB staining.


EAE was induced in GFAP - Cre OTUB1fl / fl mice ( n = 29 ) and OTUB1fl / fl control littermates ( n = 29 ) by MOG35 - 55 peptide immunization with pertussis toxin . Graph represents data pooled from 4 experiments with 7 - 8 mice per group and shows the mean clinical scores + SEM . Statistical analysis performed using Mann - Whitney U test ; * p < 0 . 05 . EAE was induced in GFAP - Cre OTUB1fl / fl mice ( n = 12 ) and OTUB1fl / fl control littermates ( n = 12 ) by MOG35 - 55 peptide immunization without pertussis toxin . Graph represents the mean clinical scores + SEM . Statistical analysis performed using Mann - Whitney U test ; * p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

EAE was induced in GFAP-Cre OTUB1fl/fl mice (n = 29) and OTUB1fl/fl control littermates (n = 29) by MOG35-55 peptide immunization with pertussis toxin. Graph represents data pooled from 4 experiments with 7-8 mice per group and shows the mean clinical scores + SEM. Statistical analysis performed using Mann-Whitney U test; * p < 0.05. EAE was induced in GFAP-Cre OTUB1fl/fl mice (n = 12) and OTUB1fl/fl control littermates (n = 12) by MOG35-55 peptide immunization without pertussis toxin. Graph represents the mean clinical scores + SEM. Statistical analysis performed using Mann-Whitney U test; * p < 0.05.


Percentages of CD4 + and CD8 + T cells infiltrating the spinal cord of OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice were analyzed by flow cytometry at day 15 ( n = 7 for OTUB1fl / fl group ; n = 9 for GFAP - Cre OTUB1fl / fl group ) and 22 ( n = 4 for OTUB1fl / fl group ; n = 5 for GFAP - Cre OTUB1fl / fl group ) p . i . Representative dot plots are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Percentages of CD4+ and CD8+ T cells infiltrating the spinal cord of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were analyzed by flow cytometry at day 15 (n = 7 for OTUB1fl/fl group; n = 9 for GFAP-Cre OTUB1fl/fl group) and 22 (n = 4 for OTUB1fl/fl group; n = 5 for GFAP-Cre OTUB1fl/fl group) p.i. Representative dot plots are shown.


Absolute number of infiltrating leukocytes in the spinal cord of OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice at day 15 ( n = 7 for OTUB1fl / fl group ; n = 9 for GFAP - Cre OTUB1fl / fl group ) and 22 ( n = 4 for OTUB1fl / fl group ; n = 5 for GFAP - Cre OTUB1fl / fl group ) p . i . was calculated based on flow cytometry results . Data show mean + SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Absolute number of infiltrating leukocytes in the spinal cord of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice at day 15 (n = 7 for OTUB1fl/fl group; n = 9 for GFAP-Cre OTUB1fl/fl group) and 22 (n = 4 for OTUB1fl/fl group; n = 5 for GFAP-Cre OTUB1fl/fl group) p.i. was calculated based on flow cytometry results. Data show mean + SEM.


Infiltrating CD4 + T cells were further analyzed by flow cytometry for their ability to produce GM - CSF , IFN - γ , and IL - 17 at day 15 and 22 p . i . , respectively . Representative dot plots are shown . Absolute number of infiltrating GM - CSF - , IFN - γ - , and IL - 17 - producing CD4 + T cells at day 15 ( n = 7 for OTUB1fl / fl group ; n = 9 for GFAP - Cre OTUB1fl / fl group ) and 22 ( n = 4 for OTUB1fl / fl group ; n = 5 for GFAP - Cre OTUB1fl / fl group ) p . i . was calculated based on flow cytometry results ( mean + SEM ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Infiltrating CD4+ T cells were further analyzed by flow cytometry for their ability to produce GM-CSF, IFN-γ, and IL-17 at day 15 and 22 p.i., respectively. Representative dot plots are shown. Absolute number of infiltrating GM-CSF-, IFN-γ-, and IL-17-producing CD4+ T cells at day 15 (n = 7 for OTUB1fl/fl group; n = 9 for GFAP-Cre OTUB1fl/fl group) and 22 (n = 4 for OTUB1fl/fl group; n = 5 for GFAP-Cre OTUB1fl/fl group) p.i. was calculated based on flow cytometry results (mean + SEM).


Relative expression of GM - CSF , IFN - γ , IL - 17 , TNF , IL - 6 , CXCL1 , CXCL10 , CXCL11 , CCL2 and NOS2 mRNA in the spinal cord of OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice at day 15 and 22 p . i . was determined by quantitative real - time PCR . Data are presented as the relative increase over unimmunized control mice ( n = 3 for all groups ) . Data show mean + SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Relative expression of GM-CSF, IFN-γ, IL-17, TNF, IL-6, CXCL1, CXCL10, CXCL11, CCL2 and NOS2 mRNA in the spinal cord of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice at day 15 and 22 p.i. was determined by quantitative real-time PCR. Data are presented as the relative increase over unimmunized control mice (n = 3 for all groups). Data show mean + SEM.


Astrocytes were isolated from unimmunized and diseased OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice at day 15 p . i . , respectively . Relative expression of CXCL10 , CXCL11 , CCL2 , and NOS2 mRNA was determined by quantitative real - time PCR . Data are presented as relative increase of genes at day 15 p . i . over gene expression of astrocytes from unimmunized control mice ( n = 3 for all groups ) . Data show mean + SEM .

Astrocytes were isolated from unimmunized and diseased OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice at day 15 p.i., respectively. Relative expression of CXCL10, CXCL11, CCL2, and NOS2 mRNA was determined by quantitative real-time PCR. Data are presented as relative increase of genes at day 15 p.i. over gene expression of astrocytes from unimmunized control mice (n = 3 for all groups). Data show mean + SEM.


Primary astrocytes isolated from OTUB1fl / fl ( n = 3 ) and GFAP - Cre OTUB1fl / fl ( n = 3 ) mice were left untreated or stimulated with IFN - γ ( 10 ng / ml ) , IL - 17 ( 50 ng / ml ) , and TNF ( 10 ng / ml ) , respectively , for 16 h . mRNA levels of CXCL10 , CXCL11 , CCL2 , and NOS2 were detected by quantitative real - time PCR .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary astrocytes isolated from OTUB1fl/fl (n = 3) and GFAP-Cre OTUB1fl/fl (n = 3) mice were left untreated or stimulated with IFN-γ (10 ng/ml), IL-17 (50 ng/ml), and TNF (10 ng/ml), respectively, for 16 h. mRNA levels of CXCL10, CXCL11, CCL2, and NOS2 were detected by quantitative real-time PCR.


Primary astrocytes from OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice were treated with IFN - γ ( 10 ng / ml ) for indicated times and analyzed by western blot with indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were treated with IFN-γ (10 ng/ml) for indicated times and analyzed by western blot with indicated antibodies.


Primary astrocytes from OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice were treated with IFN - γ ( 10 ng / ml ) for indicated times . Cytoplasmic and nuclear extracts were separated and analyzed by WB with indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were treated with IFN-γ (10 ng/ml) for indicated times. Cytoplasmic and nuclear extracts were separated and analyzed by WB with indicated antibodies.


Astrocytes were selectively isolated by MACS from OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice at days 0 and 15 p . i . , respectively . Relative expression of STAT1 mRNA was determined by quantitative real - time PCR . Data show the relative increase of STAT1 mRNA over that of unimmunized control mice ( n = 3 for all groups ) ( mean + SEM ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Astrocytes were selectively isolated by MACS from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice at days 0 and 15 p.i., respectively. Relative expression of STAT1 mRNA was determined by quantitative real-time PCR. Data show the relative increase of STAT1 mRNA over that of unimmunized control mice (n = 3 for all groups) (mean + SEM).


Primary astrocytes from OTUB1fl / fl ( n = 3 ) and GFAP - Cre OTUB1fl / fl ( n = 3 ) mice were stimulated with IFN - γ ( 10 ng / ml ) for indicated times . mRNA was isolated and analyzed by quantitative real - time PCR for SOCS1 . Data show the relative increase over untreated controls ( mean + SEM ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary astrocytes from OTUB1fl/fl (n = 3) and GFAP-Cre OTUB1fl/fl (n = 3) mice were stimulated with IFN-γ (10 ng/ml) for indicated times. mRNA was isolated and analyzed by quantitative real-time PCR for SOCS1. Data show the relative increase over untreated controls (mean + SEM).


Primary astrocytes from wild type mice were stimulated with IFN - γ ( 10 ng / ml ) for indicated times . Proteins were immunoprecipitated from whole cell lysates with anti - OTUB1 antibody and analyzed by WB with antibodies against JAK1 , JAK2 , SOCS1 , and OTUB1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary astrocytes from wild type mice were stimulated with IFN-γ (10 ng/ml) for indicated times. Proteins were immunoprecipitated from whole cell lysates with anti-OTUB1 antibody and analyzed by WB with antibodies against JAK1, JAK2, SOCS1, and OTUB1.


Primary astrocytes from OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice were left untreated or treated with IFN - γ ( 10 ng / ml ) for 30 min . Proteins were immunoprecipitated with anti - SOCS1 antibody and analyzed by WB for SOCS1 , OTUB1 , and K48 Ubiquitin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were left untreated or treated with IFN-γ (10 ng/ml) for 30 min. Proteins were immunoprecipitated with anti-SOCS1 antibody and analyzed by WB for SOCS1, OTUB1, and K48 Ubiquitin.


Primary astrocytes from GFAP - Cre OTUB1fl / fl mice were cotransfected with SOCS1 - MYC , K48 ubiquitin - HA , and OTUB1 - GFP / OTUB1 - ∆ N - GFP / OTUB1 - C91S - GFP plasmids . Twenty - four hours after transfection , cells were left untreated or stimulated with IFN - γ ( 10 ng / ml ) for 30 min before lysation . Proteins were immunoprecipitated with anti - MYC antibody and analyzed by WB for MYC , GFP , and HA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary astrocytes from GFAP-Cre OTUB1fl/fl mice were cotransfected with SOCS1-MYC, K48 ubiquitin-HA, and OTUB1-GFP/OTUB1-∆N-GFP/OTUB1-C91S-GFP plasmids. Twenty-four hours after transfection, cells were left untreated or stimulated with IFN-γ (10 ng/ml) for 30 min before lysation. Proteins were immunoprecipitated with anti-MYC antibody and analyzed by WB for MYC, GFP, and HA.


Primary astrocytes from OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice were transfected with SOCS1 - MYC plasmids . Twenty - four hours after transfection , cells were treated with cycloheximide ( CHX , 100 μg / ml ) for indicated times . SOCS1 protein levels were analyzed by WB ( upper panel ) . The lower panel shows the relative protein levels of SOCS1 normalized to Tubulin ( n = 3 for both groups ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were transfected with SOCS1-MYC plasmids. Twenty-four hours after transfection, cells were treated with cycloheximide (CHX, 100 μg/ml) for indicated times. SOCS1 protein levels were analyzed by WB (upper panel). The lower panel shows the relative protein levels of SOCS1 normalized to Tubulin (n = 3 for both groups).


Primary astrocytes from GFAP - Cre OTUB1fl / fl mice were transfected with SOCS1 - MYC plasmids or cotransfected with SOCS1 - MYC + OTUB1 - GFP plasmids . Twenty - four hours after transfection , cells were treated with CHX ( 100 μg / ml ) for indicated times . SOCS1 protein levels were analyzed by WB ( upper panel ) . The lower panel shows the relative protein levels of SOCS1 normalized to Tubulin ( n = 3 for both groups ) ( mean + SEM ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary astrocytes from GFAP-Cre OTUB1fl/fl mice were transfected with SOCS1-MYC plasmids or cotransfected with SOCS1-MYC + OTUB1-GFP plasmids. Twenty-four hours after transfection, cells were treated with CHX (100 μg/ml) for indicated times. SOCS1 protein levels were analyzed by WB (upper panel). The lower panel shows the relative protein levels of SOCS1 normalized to Tubulin (n = 3 for both groups) (mean + SEM).


Primary astrocytes from GFAP - Cre OTUB1fl / fl mice were cotransfected with SOCS1 - MYC and OTUB1 - GFP / OTUB1 - ∆ N - GFP / OTUB1 - C91S - GFP plasmids . Twenty - four hours after transfection , cells were treated with CHX ( 100 μg / ml ) for indicated times . SOCS1 protein levels were analyzed by WB ( left panel ) . The right panel shows the relative protein levels of SOCS1 normalized to Tubulin ( n = 4 for both groups ) . Data represent mean + SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary astrocytes from GFAP-Cre OTUB1fl/fl mice were cotransfected with SOCS1-MYC and OTUB1-GFP/OTUB1-∆N-GFP/OTUB1-C91S-GFP plasmids. Twenty-four hours after transfection, cells were treated with CHX (100 μg/ml) for indicated times. SOCS1 protein levels were analyzed by WB (left panel). The right panel shows the relative protein levels of SOCS1 normalized to Tubulin (n = 4 for both groups). Data represent mean + SEM.


Primary astrocytes from OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice were stimulated with IFN - γ ( 10 ng / ml ) for four hours . Thereafter , cells were treated with CHX ( 100 μg / ml ) for indicated times . Whole cell lysates were analyzed by WB with indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were stimulated with IFN-γ (10 ng/ml) for four hours. Thereafter, cells were treated with CHX (100 μg/ml) for indicated times. Whole cell lysates were analyzed by WB with indicated antibodies.


Primary astrocytes from OTUB1fl / fl mice were transfected with SOCS1 - MYC plasmids . Five hours later , astrocytes were subsequently transfected with different siRNAs or left untreated . Twenty - four hours after siRNA transfection , protein was isolated and analyzed by WB for SOCS1 expression .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary astrocytes from OTUB1fl/fl mice were transfected with SOCS1-MYC plasmids. Five hours later, astrocytes were subsequently transfected with different siRNAs or left untreated. Twenty-four hours after siRNA transfection, protein was isolated and analyzed by WB for SOCS1 expression.


Primary astrocytes from OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice were transfected with nonsense siRNA and SOCS1 siRNA - 2 . Twenty - four hours after transfection , cells were stimulated with IFN - γ ( 10 μg / ml ) for 16 hours before mRNA isolation . Quantitative real - time PCR was performed to detect mRNA levels of CXCL10 , CCL2 , and NOS2 ( n = 4 for all groups ) . Data show mean + SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were transfected with nonsense siRNA and SOCS1 siRNA-2. Twenty-four hours after transfection, cells were stimulated with IFN-γ (10 μg/ml) for 16 hours before mRNA isolation. Quantitative real-time PCR was performed to detect mRNA levels of CXCL10, CCL2, and NOS2 (n = 4 for all groups). Data show mean + SEM.


Primary astrocytes from OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice were treated with IFN - β ( 10 ng / ml ) for indicated times . Whole cell lysates were analyzed by WB with indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were treated with IFN-β (10 ng/ml) for indicated times. Whole cell lysates were analyzed by WB with indicated antibodies.


Primary astrocytes from OTUB1fl / fl ( n = 3 ) and GFAP - Cre OTUB1fl / fl ( n = 3 ) mice were treated with IFN - β ( 10 ng / ml ) for 16 hours . Quantitative real - time PCR was performed to detect OTUB1 ( B ) , CXCL10 ( C ) , CXCL11 ( D ) , CCL2 ( E ) , and NOS2 ( F ) mRNA levels . Data show mean + SEM .

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Primary astrocytes from OTUB1fl/fl (n = 3) and GFAP-Cre OTUB1fl/fl (n = 3) mice were treated with IFN-β (10 ng/ml) for 16 hours. Quantitative real-time PCR was performed to detect OTUB1 (B), CXCL10 (C), CXCL11 (D), CCL2 (E), and NOS2 (F) mRNA levels. Data show mean + SEM.


Sucrose gradient UV profile of 60S / 40S / IAPV - IRES reaction mixture after an overnight run . The peaks corresponding to 80S and 40S were used for RNA extraction and UREA - PAGE shown in the inset .

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Sucrose gradient UV profile of 60S/40S/IAPV-IRES reaction mixture after an overnight run. The peaks corresponding to 80S and 40S were used for RNA extraction and UREA-PAGE shown in the inset.


Representative cryo - EM image where roughly half of the particles correspond to 40S ( blue ) and the other half to 80S ( orange ) .

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Representative cryo-EM image where roughly half of the particles correspond to 40S (blue) and the other half to 80S (orange).


Size exclusion chromatography profiles of recombinant proteins used in this study . AU : arbitrary units ; WT : wild type ; FL : full - length .

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Size exclusion chromatography profiles of recombinant proteins used in this study. AU: arbitrary units; WT: wild type; FL: full-length.


Analysis of the interaction between CtUba4 and GST - CtUrm1 ( ratio 2 : 1 or 0 . 5 : 1 when marked with an asterisk ) in the presence of nucleotide derivates by GST pull - down resolved by SDS - PAGE and visualized with Coomassie stain .

Analysis of the interaction between CtUba4 and GST-CtUrm1 (ratio 2:1 or 0.5:1 when marked with an asterisk) in the presence of nucleotide derivates by GST pull-down resolved by SDS-PAGE and visualized with Coomassie stain.


Functional analyses of ScUba4 mutant yeast using APM - gel retardation ( top ) and viability in response to rapamycin ( bottom ) . PAGE was supplemented with APM to retard the migration of thiolated tRNAs and allow their visualization by northern blot using an anti - tRNA < sub > UU < em > C < / em > < / sub > < sup > Gl < em > u < / em > < / sup > ; probe . APM : ( [ N - Acryloyl - amino ] phenyl ) mercuric chloride ; YPD : yeast extract peptone dextrose . All residue numbering follows the CtUba4 sequence , but the respective ScUba4 numbering is added in subscript .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Functional analyses of ScUba4 mutant yeast using APM-gel retardation (top) and viability in response to rapamycin (bottom). PAGE was supplemented with APM to retard the migration of thiolated tRNAs and allow their visualization by northern blot using an anti-tRNAUUCGlu; probe. APM: ([N-Acryloyl-amino]phenyl)mercuric chloride; YPD: yeast extract peptone dextrose. All residue numbering follows the CtUba4 sequence, but the respective ScUba4 numbering is added in subscript.


Interaction analysis of WT and mutated CtUba4 with GST - CtUrm1 by GST pull - down in the presence of 1mM ATP . CtUba4C202K covalently linked to GST - CtUrm1 is marked by an asterisk .

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Interaction analysis of WT and mutated CtUba4 with GST-CtUrm1 by GST pull-down in the presence of 1mM ATP. CtUba4C202K covalently linked to GST-CtUrm1 is marked by an asterisk.


Analysis of covalent adduct formation between CtUba4 WT or C202 mutants and carboxylated ( - OH ) or thiocarboxylated ( - SH ) CtUrm1 in the presence or absence of TBH , DTT , TCEP and HA , respectively . ATP : Adenosine triphosphate TBH : tert - Butyl hydroperoxide HA : Hydroxylamine ; DTT : 1 , 4 - Dithiothreitol ; TCEP : Tris ( 2 - carboxyethyl ) phosphine .

Analysis of covalent adduct formation between CtUba4 WT or C202 mutants and carboxylated (-OH) or thiocarboxylated (-SH) CtUrm1 in the presence or absence of TBH, DTT, TCEP and HA, respectively. ATP: Adenosine triphosphate TBH: tert-Butyl hydroperoxide HA: Hydroxylamine; DTT: 1,4-Dithiothreitol; TCEP: Tris(2-carboxyethyl)phosphine.


Analysis of covalent adduct formation between CtUba4 WT / cysteine mutants and CtUrm1 - COSH in the presence of oxidizing agents . HP : Hydrogen peroxide ; D : Diamide .

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Analysis of covalent adduct formation between CtUba4 WT/cysteine mutants and CtUrm1-COSH in the presence of oxidizing agents. HP: Hydrogen peroxide; D: Diamide.


Phenotypic analysis of ScUba4 mutant yeast strains in response to rapamycin .

Phenotypic analysis of ScUba4 mutant yeast strains in response to rapamycin.


MS / MS spectrum of the K202 - ε - GGH containing peptide from CtUba4C202K . A representative annotated fragmentation spectrum is shown with the b - and y - ions marked in red and blue , respectively . The precursor ion at m / z 591 , 98 is labelled in green . The peptide sequence is shown at the top with the collision - induced fragmentation pattern . The Urm1 conjugation site was identified by detection of the GGH remnant motif generated by chymotryptic digestion .

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MS/MS spectrum of the K202-ε-GGH containing peptide from CtUba4C202K. A representative annotated fragmentation spectrum is shown with the b- and y-ions marked in red and blue, respectively. The precursor ion at m/z 591,98 is labelled in green. The peptide sequence is shown at the top with the collision-induced fragmentation pattern. The Urm1 conjugation site was identified by detection of the GGH remnant motif generated by chymotryptic digestion.


Analysis of covalent adduct formation between CtUba4 WT or Cys202 mutants and thiocarboxylated ( - SH ) CtUrm1 in the presence or absence of TBH and HA , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O

Analysis of covalent adduct formation between CtUba4 WT or Cys202 mutants and thiocarboxylated (-SH) CtUrm1 in the presence or absence of TBH and HA, respectively.


B . Quantification of time - course nucleosome H2A - Ub assays using the indicated BRCA1 / BARD1 truncations . Data are presented as the normalized fraction of H2A consumed during a time - course nucleosome ubiquitylation assay . Data points represent mean values and error bars are ± 1 - s . d . of n = 3 independent technical replicate experiments . The residue bounds of BRCA1RING are 1 - 112 , and BARD1RING are 26 - 140 . Additional details about all reagents used in these studies can be found in the methods section .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Quantification of time-course nucleosome H2A-Ub assays using the indicated BRCA1/BARD1 truncations. Data are presented as the normalized fraction of H2A consumed during a time-course nucleosome ubiquitylation assay. Data points represent mean values and error bars are ± 1-s.d. of n=3 independent technical replicate experiments. The residue bounds of BRCA1RING are 1-112, and BARD1RING are 26-140. Additional details about all reagents used in these studies can be found in the methods section.


C . Nucleosome binding curves from fluorescence - quenching - based measurements using the indicated BRCA1 / BARD1 constructs . Data show mean values and error bars are ± 1 - s . d . of n = 4 independent technical replicate experiments . The reported difference in affinities to BRCA1RING / BARD1RING is likely underestimated , as the minimal RING / RING heterodimer binding data was collected at a lower ionic strength than the other constructs to obtain well - behaved binding data ( 50 mM vs . 100 mM NaCl ) .

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C. Nucleosome binding curves from fluorescence-quenching-based measurements using the indicated BRCA1/BARD1 constructs. Data show mean values and error bars are ± 1-s.d. of n=4 independent technical replicate experiments. The reported difference in affinities to BRCA1RING/BARD1RING is likely underestimated, as the minimal RING/RING heterodimer binding data was collected at a lower ionic strength than the other constructs to obtain well-behaved binding data (50 mM vs. 100 mM NaCl).


B . 1H15N - HSQC NMR amide chemical shift perturbations ( CSPs ) observed for 15N - BARD1 141 - 216 signals when bound to a 36 - bp dsDNA fragment ( 1 : 1 molar equivalent complex ) .

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B. 1H15N-HSQC NMR amide chemical shift perturbations (CSPs) observed for 15N-BARD1 141-216 signals when bound to a 36-bp dsDNA fragment (1:1 molar equivalent complex).


C . Representative SDS - PAGE gel monitoring in - gel fluorescence ( labelled DNA ) of UV - induced Bpa crosslinking of the E3 ligase fusion BRCA1 - ƒ - BARD1221 ( BCƒBD ) to nucleosomes ( left ) . In this construct , the C - terminus of BRCA11 - 104 is genetically fused to the N - terminus of BARD126 - 221 via a 12 - residue GlySer - linker . Quantification of Bpa crosslinking experiments with nucleosomes ( right ) . The intensity of each crosslinked band was normalized to the intensity of the L120Bpa crosslinked band for each replicate experiment . Graph bars show the mean ; error bars are ± 1 - s . d . and the open circles are the values of individual replicates for of n = 3 independent technical replicate experiments .

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C. Representative SDS-PAGE gel monitoring in-gel fluorescence (labelled DNA) of UV-induced Bpa crosslinking of the E3 ligase fusion BRCA1-ƒ-BARD1221 (BCƒBD) to nucleosomes (left). In this construct, the C-terminus of BRCA11-104 is genetically fused to the N-terminus of BARD126-221 via a 12-residue GlySer-linker. Quantification of Bpa crosslinking experiments with nucleosomes (right). The intensity of each crosslinked band was normalized to the intensity of the L120Bpa crosslinked band for each replicate experiment. Graph bars show the mean; error bars are ± 1-s.d. and the open circles are the values of individual replicates for of n=3 independent technical replicate experiments.


Quantification of H2A - Ub time - course assays using the indicated BRCA1 / BARD1 constructs . Data show the mean ; error bars are ± 1 - s . d . of n = 3 independent technical replicate experiments .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of H2A-Ub time-course assays using the indicated BRCA1/BARD1 constructs. Data show the mean; error bars are ± 1-s.d. of n=3 independent technical replicate experiments.


Two single time - points from panel C from the BRCA1RING / BARD1RING curve were excluded from analysis due to their extremely low activities coupled with quenching / loading errors yielding normalized fraction of H2A consumed values of < 0 . Values for these excluded points are included as source data .

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Two single time-points from panel C from the BRCA1RING/BARD1RING curve were excluded from analysis due to their extremely low activities coupled with quenching/loading errors yielding normalized fraction of H2A consumed values of <0. Values for these excluded points are included as source data.


C . Inhibition of UV - induced Bpa crosslinking between the indicated BRCA1 - f - BARD1221 Bpa - incorporated constructs and NCP185 substrates in the presence of increasing amounts of dsDNA or bubble - DNA competitor . Data show the mean ; error bars are ± 1 - s . d . of n = 3 ( W146Bpa ) or n = 4 ( A195Bpa ) independent technical replicate experiments .

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C. Inhibition of UV-induced Bpa crosslinking between the indicated BRCA1-f-BARD1221 Bpa-incorporated constructs and NCP185 substrates in the presence of increasing amounts of dsDNA or bubble-DNA competitor. Data show the mean; error bars are ± 1-s.d. of n=3 (W146Bpa) or n=4 (A195Bpa) independent technical replicate experiments.


D . 1H15N - HSQC NMR CSPs observed to 15N - BARD1 141 - 216 signals when bound to a 36 - mer dsDNA ( blue bars ) or bubble - DNA ( gray bars with black outlines ) fragment ( 1 : 1 molar equivalent complex ) . Signals for residues 143 - 157 are broadened beyond detection in the bubble - DNA - bound spectrum ; for visualization purposes , gray bars are set equal to the CSP value for the W146Ne CSP that was observed ; the real CSP values are likely larger that this

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D. 1H15N-HSQC NMR CSPs observed to 15N-BARD1 141-216 signals when bound to a 36-mer dsDNA (blue bars) or bubble-DNA (gray bars with black outlines) fragment (1:1 molar equivalent complex). Signals for residues 143-157 are broadened beyond detection in the bubble-DNA-bound spectrum; for visualization purposes, gray bars are set equal to the CSP value for the W146Ne CSP that was observed; the real CSP values are likely larger that this value


F . Single time - point H2A - Ub inhibition assays using heterodimers containing BRCA1RING / BARD1FL ( left , 12 min endpoint ) or the indicated BARD1 internal deletion mutant ( middle and right , 20 min endpoint ) and increasing amounts of dsDNA or bubble - DNA competitor . The same E3 concentration ( 50 nM ) was used for each BRCA1 / BARD1 truncation . A longer time point was used for the double - deletion mutant as the intrinsic H2A - Ub activity of this mutant was lower , likely due to the deletion of DNA binding regions .

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F. Single time-point H2A-Ub inhibition assays using heterodimers containing BRCA1RING/BARD1FL (left, 12 min endpoint) or the indicated BARD1 internal deletion mutant (middle and right, 20 min endpoint) and increasing amounts of dsDNA or bubble-DNA competitor. The same E3 concentration (50 nM) was used for each BRCA1/BARD1 truncation. A longer time point was used for the double-deletion mutant as the intrinsic H2A-Ub activity of this mutant was lower, likely due to the deletion of DNA binding regions.


A . Western blot analysis to detect HA - BARD1 in nuclei isolated from HeLa - shBARD1 cells stably expressing wild - type or Δ194 - 216 mutant of HA - BARD1 , where endogenous BARD1 was depleted by doxycycline - induced shBARD1 expression . The nuclei were salt - fractionated into NS100 , NS300 , NS420 , and NP420 to assess the amount of HA - BARD1 associated with chromatin . Data are representative of n = 2 biological replicate experiments .

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A. Western blot analysis to detect HA-BARD1 in nuclei isolated from HeLa-shBARD1 cells stably expressing wild-type or Δ194-216 mutant of HA-BARD1, where endogenous BARD1 was depleted by doxycycline-induced shBARD1 expression. The nuclei were salt-fractionated into NS100, NS300, NS420, and NP420 to assess the amount of HA-BARD1 associated with chromatin. Data are representative of n=2 biological replicate experiments.


B . Quantification of cells with > 5 HA - BARD1 foci ( left panel ) and number of HA - BARD1 foci per nucleus ( right panel ) with and without olaparib treatment . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , by Student ' s t - test . Data show the individual points from n = 3 biological replicate experiments .

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B. Quantification of cells with >5 HA-BARD1 foci (left panel) and number of HA-BARD1 foci per nucleus (right panel) with and without olaparib treatment. *p<0.05, **p<0.01, ***p<0.001, by Student's t-test. Data show the individual points from n=3 biological replicate experiments.


C . Clonogenic survival of HeLa - shBARD1 cells stably expressing wild type or Δ194 - 216 mutant of HA - BARD1 upon treatment with indicated amount of olaparib and cisplatin . Data points show the mean ; error bars represent SEM of n = 3 biological replicate experiments .

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C. Clonogenic survival of HeLa-shBARD1 cells stably expressing wild type or Δ194-216 mutant of HA-BARD1 upon treatment with indicated amount of olaparib and cisplatin. Data points show the mean; error bars represent SEM of n=3 biological replicate experiments.


B , C . Quantification of time - course H2A - Ub assays using the indicated BRCA1 / BARD1 constructs and chromatin substrates . data show the mean ; error bars are ± 1 - s . d . of n = 3 independent technical replicate experiments .

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B, C. Quantification of time-course H2A-Ub assays using the indicated BRCA1/BARD1 constructs and chromatin substrates. data show the mean; error bars are ± 1-s.d. of n=3 independent technical replicate experiments.


D . Quantification of time - course H2A - Ub assays using homogenously modified H2A K15 - Ub mono - nucleosome substrates and indicated BRCA1 / BARD1 constructs . data show the mean ; error bars are ± 1 - s . d . of n = 3 independent technical replicate experiments . One time - point from Panel D was determined to be an outlier due to a reaction quenching error and excluded from analysis . The excluded value is reported as source data .

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D. Quantification of time-course H2A-Ub assays using homogenously modified H2A K15-Ub mono-nucleosome substrates and indicated BRCA1/BARD1 constructs. data show the mean; error bars are ± 1-s.d. of n=3 independent technical replicate experiments. One time-point from Panel D was determined to be an outlier due to a reaction quenching error and excluded from analysis. The excluded value is reported as source data.


G , H . Quantification of time - course H2A - Ub assays using the indicated combinations of di - NCP substrate and BRCA1 / BARD1 heterodimer . In each case , the y - axis reports on the fraction of H2Aobserve ( as shown in Panel F ) . Lower E3 concentrations were used in these reactions than in those presented in Figure 1 using unmodified mono - nucleosome substrates ( 15 nM vs . 100 nM ) , accounting for the slower observed H2A - Ub kinetics for the unmodified di - nucleosome substrate . data show the mean ; error bars are ± 1 - s . d . of n = 3 independent technical replicate experiments .

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G, H. Quantification of time-course H2A-Ub assays using the indicated combinations of di-NCP substrate and BRCA1/BARD1 heterodimer. In each case, the y-axis reports on the fraction of H2Aobserve (as shown in Panel F). Lower E3 concentrations were used in these reactions than in those presented in Figure 1 using unmodified mono-nucleosome substrates (15 nM vs. 100 nM), accounting for the slower observed H2A-Ub kinetics for the unmodified di-nucleosome substrate. data show the mean; error bars are ± 1-s.d. of n=3 independent technical replicate experiments.


A - I . Correlative light electron microscopy ( CLEM ) showing Prom1 at the plasma membrane of primary hepatocytes . Primary hepatocytes were isolated from 12 - week - old male WT ( Prom1 + / + ) or KO ( Prom1cre - ert2 - nlacz ) mice . The boxed areas in panel ( A , D , G ) and ( B , E , H ) were magnified and presented as panel ( B , E , H ) and ( C , F , I ) , respectively . Scale bar ; 5 μm ( A , D and G ) , 1 μm ( B , E and H ) , and 0 . 2 μm ( C , F and I ) .

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A-I. Correlative light electron microscopy (CLEM) showing Prom1 at the plasma membrane of primary hepatocytes. Primary hepatocytes were isolated from 12-week-old male WT (Prom1+/+) or KO ( Prom1cre-ert2-nlacz) mice. The boxed areas in panel (A, D, G) and (B, E, H) were magnified and presented as panel (B, E, H) and (C, F, I), respectively. Scale bar; 5 μm (A, D and G), 1 μm (B, E and H), and 0.2 μm (C, F and I).


J . Immunofluorescent staining of Prom1 , CK19 and HNF4α in two serial cryosections of mouse liver . The boxed area in the top panel was magnified and presented as the bottom panel . Asterisks ( * ) in the top panel indicate cholangiocytes of the bile bucts and triangles ( Γ ) in the bottom panel indicate hepatocytes . Scale bar , 100 μm .

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J. Immunofluorescent staining of Prom1, CK19 and HNF4α in two serial cryosections of mouse liver. The boxed area in the top panel was magnified and presented as the bottom panel. Asterisks (*) in the top panel indicate cholangiocytes of the bile bucts and triangles (Γ) in the bottom panel indicate hepatocytes. Scale bar, 100 μm.


Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice and serum - starved . ( K ) Differentially expressed genes showing more than a 2 - fold change with P < 0 . 05 ( 118 DEGs ) induced ( n = 55 ) or repressed ( n = 63 ) by Prom1 knockout ( n = 2 / group ) . G6pc , glucose - 6 - phosphatase catalytic subunit ; Pck1 , phosphoenolpyruvate carboxykinase 1 .

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Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice and serum-starved. (K) Differentially expressed genes showing more than a 2-fold change with P < 0.05 (118 DEGs) induced (n=55) or repressed (n=63) by Prom1 knockout (n = 2/group). G6pc, glucose-6-phosphatase catalytic subunit; Pck1, phosphoenolpyruvate carboxykinase 1.


Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice and serum - starved . ( L ) Relative expression levels of G6pc and Pck1 mRNAs after serum starvation for 16 hours were determined by qRT - PCR ( n = 3 / group ) .

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Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice and serum-starved. (L) Relative expression levels of G6pc and Pck1 mRNAs after serum starvation for 16 hours were determined by qRT-PCR (n = 3/group).


Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice and serum - starved . ( M ) Levels of the Prom1 , PCK , G6PC and GAPDH proteins were determined by immunoblotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice and serum-starved. (M) Levels of the Prom1, PCK, G6PC and GAPDH proteins were determined by immunoblotting.


Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice and serum - starved . ( N ) Prom1 + / + or Prom1 - / - hepatocytes serum - starved for 8 hours were incubated with 2 - [ 3H ] deoxyglucose for 10 minutes . Glucose uptake was assessed by calculating the amount of cell - associated radioactivity normalized to the amount of protein ( n = 3 mice / group ) .

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Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice and serum-starved. (N) Prom1+/+ or Prom1-/- hepatocytes serum-starved for 8 hours were incubated with 2-[3H]deoxyglucose for 10 minutes. Glucose uptake was assessed by calculating the amount of cell-associated radioactivity normalized to the amount of protein (n = 3 mice/group).


A . Levels of G6pc and Pck1 mRNAs in the liver of mice after 18 hours fasting were determined by qRT - PCR . The level of each mRNA was normalized to the level of 18s rRNA ( n = 3 / fed , 7 or 8 / fasted ) .

A. Levels of G6pc and Pck1 mRNAs in the liver of mice after 18 hours fasting were determined by qRT-PCR. The level of each mRNA was normalized to the level of 18s rRNA (n = 3/fed, 7 or 8/fasted).


B . Levels of phospho - PKA substrates ( p - PKA substrates ) , phospho - CREB ( p - CREB ) , CREB , G6pc Pck , Prom1 and GAPDH were determined after 18 hours fasting . Band intensity for p - CREB was quantified using Photoshop software .

B. Levels of phospho-PKA substrates (p-PKA substrates), phospho-CREB (p-CREB), CREB, G6pc Pck, Prom1 and GAPDH were determined after 18 hours fasting. Band intensity for p-CREB was quantified using Photoshop software.


C . Blood glucose levels ( mg / dL ) of mice after fasted for 24 hours ( n = 12 or 14 / group ) .

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C. Blood glucose levels (mg/dL) of mice after fasted for 24 hours (n = 12 or 14/group).


D . Serum glucagon level in the fed or fasted mice ( n = 3 / fed , 7 or 8 / fasted ) .

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D. Serum glucagon level in the fed or fasted mice (n = 3/fed, 7 or 8/fasted).


E . Glycogen contents in the liver of mouse in each condition was shown by PAS ( periodic acid schiff ) staining . Scale bar , 500 μm .

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E. Glycogen contents in the liver of mouse in each condition was shown by PAS (periodic acid schiff) staining. Scale bar, 500 μm.


Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice , serum - starved and stimulated with glucagon . F . Levels of the G6pc and Pck1 mRNAs 2 hours after glucagon stimulation ( 10 nM ) were determined by qRT - PCR . The level of each mRNA was normalized to the level of the 18S rRNA . For these experiments , primary hepatocytes were cultured under 3D conditions using a Matrigel matrix ( n = 3 mice / group ) .

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Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. F. Levels of the G6pc and Pck1 mRNAs 2 hours after glucagon stimulation (10 nM) were determined by qRT-PCR. The level of each mRNA was normalized to the level of the 18S rRNA. For these experiments, primary hepatocytes were cultured under 3D conditions using a Matrigel matrix (n = 3 mice/group).


Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice , serum - starved and stimulated with glucagon . G . Levels of phospho - PKA substrates ( p - PKA substrates ) , phospho - CREB ( p - CREB ) , CREB , phospho - Inositol trisphosphate receptor ( p - IP3R ) , IP3R , phospho - Hormone - Sensitive Lipase ( p - HSL ) , HSL , Prom1 and Gapdh 10 minutes after glucagon stimulation ( 10 nM ) were determined by immunoblotting .

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Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. G. Levels of phospho-PKA substrates (p-PKA substrates), phospho-CREB (p-CREB), CREB, phospho-Inositol trisphosphate receptor (p-IP3R), IP3R, phospho-Hormone-Sensitive Lipase (p-HSL), HSL, Prom1 and Gapdh 10 minutes after glucagon stimulation (10 nM) were determined by immunoblotting.


Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice , serum - starved and stimulated with glucagon . H . The nuclear localization of p - CREB 10 minutes after glucagon stimulation ( 10 nM ) was determined by immunofluorescence staining . Scale bar , 20 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. H. The nuclear localization of p-CREB 10 minutes after glucagon stimulation (10 nM) was determined by immunofluorescence staining. Scale bar, 20 μm.


Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice , serum - starved and stimulated with glucagon . I . Relative PKA activities 10 minutes after glucagon stimulation ( 10 nM ) were determined using the PKA assay kit in the absence or presence of 10 μM IBMX ( n = 3 mice / group ) .

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Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. I. Relative PKA activities 10 minutes after glucagon stimulation (10 nM) were determined using the PKA assay kit in the absence or presence of 10 μM IBMX (n = 3 mice/group).


Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice , serum - starved and stimulated with glucagon . J . The cAMP concentration 10 minutes after glucagon stimulation ( 10 nM ) was determined using the cAMP assay kit in the absence or presence of 10 μM IBMX ( n = 3 mice / group ) .

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Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. J. The cAMP concentration 10 minutes after glucagon stimulation (10 nM) was determined using the cAMP assay kit in the absence or presence of 10 μM IBMX (n = 3 mice/group).


12 - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and intraperitoneally injected with glucagon . ( A ) Blood glucose levels ( mg / dL ) were measured 0 , 15 , 30 , 60 and 120 minutes after glucagon stimulation ( 200 µg / kg body weight , n = 10 / group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with glucagon. (A) Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon stimulation (200 µg/kg body weight, n = 10/group).


12 - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and intraperitoneally injected with glucagon . ( B ) Levels of p - PKA substrates , p - CREB , CREB and GAPDH in the liver after a 5 minutes glucagon treatment were determined by immunoblotting ( 2 mg / kg body weight ) .

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12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with glucagon. (B) Levels of p-PKA substrates, p-CREB, CREB and GAPDH in the liver after a 5 minutes glucagon treatment were determined by immunoblotting (2 mg/kg body weight).


12 - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and intraperitoneally injected with glucagon . ( C ) Relative PKA activities in the liver after a 5 minutes glucagon treatment were determined using the PKA assay kit ( 2 mg / kg body weight , n = 3 / group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with glucagon. (C) Relative PKA activities in the liver after a 5 minutes glucagon treatment were determined using the PKA assay kit (2 mg/kg body weight, n = 3/group).


12 - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and intraperitoneally injected with glucagon . ( D ) The cAMP concentration in the liver after a 5 minutes glucagon treatment was determined using the cAMP assay kit ( 2 mg / kg body weight , n = 3 / group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with glucagon. (D) The cAMP concentration in the liver after a 5 minutes glucagon treatment was determined using the cAMP assay kit (2 mg/kg body weight, n = 3/group).


Glucose disposal rates were measured in 12 - week - old male mice using glucose , pyruvate ( E ) tolerance tests ( n = 10 mice / group ) .

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Glucose disposal rates were measured in 12-week-old male mice using glucose, pyruvate (E) tolerance tests (n = 10 mice/group).


Glucose disposal rates were measured in 12 - week - old male mice using insulin ( F ) tolerance tests ( n = 10 mice / group ) .

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Glucose disposal rates were measured in 12-week-old male mice using insulin (F) tolerance tests (n = 10 mice/group).


G . Levels of p - AKT , AKT , p - ERK , ERK , IRβ and GAPDH were determined by immunoblotting after insulin stimulation ( 10 nM ) .

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G. Levels of p-AKT, AKT, p-ERK, ERK, IRβ and GAPDH were determined by immunoblotting after insulin stimulation (10 nM).


Primary hepatocytes were isolated from 12 - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice , serum - starved for 16 hours and stimulated with isoprenaline . ( H ) The nuclear localization of p - CREB after isoprenaline stimulation ( 10 μM ) for 10 minutes was determined by immunofluorescence staining . Scale bar , 20 μm .

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Primary hepatocytes were isolated from 12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice, serum-starved for 16 hours and stimulated with isoprenaline. (H) The nuclear localization of p-CREB after isoprenaline stimulation (10 μM) for 10 minutes was determined by immunofluorescence staining. Scale bar, 20 μm.


Primary hepatocytes were isolated from 12 - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice , serum - starved for 16 hours and stimulated with isoprenaline . ( I ) Levels of p - PKA substrates , p - CREB , CREB , Prom1 and GAPDH after isoprenaline stimulation ( 10 μM ) for 0 , 10 or 20 minutes were determined by immunoblotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

Primary hepatocytes were isolated from 12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice, serum-starved for 16 hours and stimulated with isoprenaline. (I) Levels of p-PKA substrates, p-CREB, CREB, Prom1 and GAPDH after isoprenaline stimulation (10 μM) for 0, 10 or 20 minutes were determined by immunoblotting.


Primary hepatocytes were isolated from 12 - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice , serum - starved for 16 hours and stimulated with isoprenaline . ( J ) The cAMP concentration after isoprenaline stimulation ( 10 μM ) for 10 minutes was determined using the cAMP assay kit in the presence of 10 μM IBMX ( n = 3 / group ) .

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Primary hepatocytes were isolated from 12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice, serum-starved for 16 hours and stimulated with isoprenaline. (J) The cAMP concentration after isoprenaline stimulation (10 μM) for 10 minutes was determined using the cAMP assay kit in the presence of 10 μM IBMX (n = 3/group).


WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and intraperitoneally injected with epinephrine ( 3µg / 10g ) . ( K ) Blood glucose levels ( mg / dL ) were measured 0 , 15 , 30 , 60 and 120 minutes after epinephrine

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WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with epinephrine (3µg/10g). (K) Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after epinephrine stimulation


WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and intraperitoneally injected with epinephrine ( 3µg / 10g ) . ( L ) Levels of p - PKA substrates , p - CREB , CREB and GAPDH in the liver 15 minutes after epinephrine treatment were determined by immunoblotting .

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WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with epinephrine (3µg/10g). (L) Levels of p-PKA substrates, p-CREB, CREB and GAPDH in the liver 15 minutes after epinephrine treatment were determined by immunoblotting.


M - O . WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and subjected to immobilization test . ( M ) Blood glucose levels ( mg / dL ) were measured 0 , 60 and 120 minutes after immobilization ( n = 10 / group ) .

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M-O. WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and subjected to immobilization test. (M) Blood glucose levels (mg/dL) were measured 0, 60 and 120 minutes after immobilization (n = 10/group).


WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and subjected to immobilization test . ( N ) Levels of p - PKA substrates , p - CREB , CREB and GAPDH in the liver after a 30 minutes - immobilization were determined by immunoblotting .

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WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and subjected to immobilization test. (N) Levels of p-PKA substrates, p-CREB, CREB and GAPDH in the liver after a 30 minutes-immobilization were determined by immunoblotting.


WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and subjected to immobilization test . ( O ) Serum epinephrine level of mice after a 30 minutes - immobilization was determined ( n = 3 / control group , 7 or 8 / immobilized group ) .

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WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and subjected to immobilization test. (O) Serum epinephrine level of mice after a 30 minutes-immobilization was determined (n = 3/control group, 7 or 8/immobilized group).


A . The expression of Prom1 , radixin , AKAP7 , AKAP8 , AKAP8I , AKAP9 , AKAP10 , AKAP12 or AKAP13 was silenced in WT hepatocytes using siRNAs . Hepatocytes were then serum - starved for 18 hours and stimulated with glucagon ( 10 nM ) for 10 minutes . The phosphorylation of PKA substrates was determined by immunoblotting .

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A. The expression of Prom1, radixin, AKAP7, AKAP8, AKAP8I, AKAP9, AKAP10, AKAP12 or AKAP13 was silenced in WT hepatocytes using siRNAs. Hepatocytes were then serum-starved for 18 hours and stimulated with glucagon (10 nM) for 10 minutes. The phosphorylation of PKA substrates was determined by immunoblotting.