Fig. 6

UBXN7 regulates SKIL degradation in a UAS-dependent manner. a SKIL increases in the absence of UBXN7. Nuclear extracts from parental U2OS and U2OS UBXN7-KO clones #1 and #2 treated or not with TGF-β for 1 h were analyzed by western blotting. Quantification of SKIL protein level variations is indicated below the blots panel. Values represent the mean ± SEM of SKIL intensity normalized to PARP loading control intensity and to the U2OS control condition in three independent experiments. Statistical analysis was performed using one-way ANOVA with Dunnett’s post test. ns not significant; *p < 0.05. The arrow indicates the band corresponding to RNF111. b TGF-β-induced SMAD-dependent transcription is attenuated in the absence of UBXN7. Parental U2OS and U2OS UBXN7-KO clones #1 and #2 were co-transfected with the CAGA₁₂-Luc and pRL-TK reporters and treated or not with TGF-β for 8 h before lysis. Data represent means ± SEM of luciferase activities normalized to Renilla in three independent experiments. Statistical analysis was performed using two-way ANOVA with Bonferroni post test on three independent experiments. **p < 0.01, ***p < 0.001. c UBXN7 overexpression impairs SKIL degradation in response to TGF-β. Nuclear extracts from U2OS UBXN7-KO clone #1 cells transfected with HA-tagged empty vector (-), UBXN7-WT, UBXN7-∆UAS, or UBXN7-UAS and treated or not for 1 h with TGF-β before extraction were analyzed by western blotting. Quantification and statistics as in a except that values were normalized to the empty vector condition. d UBXN7 significantly attenuates TGF-β-induced SMAD-dependent transcription in a UAS-dependent manner. U2OS UBXN7-KO clone #1 cells were co-transfected with CAGA₁₂-Luc, pRL-TK, and HA-tagged empty vector ( −) or the indicated HA-UBXN7 constructs. Luciferase assays were performed as in b. e UBXN7 inhibits SKIL ubiquitylation by RNF111. U2OS UBXN7-KO clone #1 cells were transiently transfected as indicated with HA-SKIL, Flag-RNF111-WT, Flag-RNF111-CA, and untagged UBXN7-WT. Lysates (input) were immunoprecipitated with the UB pan selector resin and subsequently analyzed by western blotting. Western blot quantification and luciferase assay values obtained for each experiments are shown in Additional file 3: Table S1