Fig. 5: The CTDNEP1-NEP1R1 phosphatase and MAN1 suppress inappropriate SMAD signaling.
From: Suppression of TGF-β/SMAD signaling by an inner nuclear membrane phosphatase complex
A Localization of endogenous SMAD2 in HeLa parental cells or lacking the indicated genes analyzed by immunofluorescence. DNA was labeled with 4′,6- diamidino- 2- phenylindole (DAPI). Scale bar: 10 μM. B Quantification of nuclear accumulation of SMAD2 from imaging experiments, as shown in (A), n = 3 independent experiments, p-values were indicated in the graph. One-way ANOVA (multiple comparison) was performed, and data are presented as mean values ± SD. C Levels of endogenous pSMAD2 and SMAD2 in HeLa parental cells or lacking the indicated genes. Cell lysates were analyzed by SDS-PAGE followed by immunoblotting with the indicated antibodies. GAPDH was used as a loading control. D Levels of endogenous pSMAD2 and SMAD2 in HeLa parental cells or CTDENP1 KO cells with indicated treatments. The 1D11 antibody against TGF-β cytokine was used at 30, 150, and 300 µg/mL; mouse IgG antibody was used at 300 µg/mL; the TGF-β receptor specific inhibitor SB431542 was used at 10 µM. Cell lysates were analyzed by SDS-PAGE followed by immunoblotting with the indicated antibodies. GAPDH was used as a loading control. E levels of p21, p15 transcripts in HeLa parental cells or lacking the indicated genes analyzed by RT-qPCR. n = 3 independent experiments, each with 3 technical replicates. p-values were indicated in the graph. One-way ANOVA (multiple comparison) was performed, and data are presented as mean values ± SD. F levels of endogenous p21 in HeLa parental cells or lacking the indicated genes. Cell lysates were analyzed by SDS-PAGE followed by immunoblotting with the indicated antibodies. GAPDH was used as a loading control. G Quantification of S phase cells assessed based on EdU incorporation and DAPI staining. At least 30000 cells were analyzed for each condition. n = 3 independent experiments, p-values were indicated in the graph. One-way ANOVA (multiple comparison) was performed, and data are presented as mean values ± SD. H The CTDNEP1-NEP1R1-MAN1 complex dephosphorylates and inactivates R-SMADs at the INM (see text for details).
