Fig. 3

UBXN7 stabilizes RNF111 in a RING and UAS dependent-manner. a Endogenous RNF111 is degraded by the proteasome in a RING-dependent manner. Western blotting of lysates from U2OS cells and CRISPR U2OS RNF111-RING-KO clones #1 and #2 treated or not with MG132 for 4 h. Quantification of variations in RNF111 protein level is indicated below the blots panel. Values represent the mean ± SEM of RNF111 intensity normalized to GAPDH loading control intensity and to the indicated control condition from three independent experiments. Statistical analysis was performed using a paired t-test. **p < 0.01, ns not significant. Arrows on western blots indicate the band corresponding to RNF111. b UBXN7 stabilizes RNF111 in a RING and UAS-dependent manner. Lysates from U2OS cells or U2OS RNF111-RING-KO clones #1 or #2 transiently transfected with HA-tagged empty vector (-), UBXN7-WT (WT), UBXN7-∆UAS (∆UAS), or UBXN7-UAS domain (UAS) were analyzed by western blotting with the indicated antibodies. Quantification of variations in RNF111 protein level was made as in a except it was normalized to the empty vector. Statistical analysis was performed using one-way ANOVA with Dunnett’s post test. *p < 0.05, **p < 0.01, ***p < 0.001, ns not significant. c Whole-cell extracts of U2OS cells transfected with a control siRNA or 2 independent siRNA #1 and #2 targeting UBXN7 were analyzed by western blotting as indicated. Quantification and statistical analysis as in b except that values were normalized to siRNA control. d Western blotting analysis of lysates from U2OS cells and U2OS UBXN7-KO clones #1 and #2 treated or not with MG132. Quantification and statistical analysis as in b except that values were normalized to U2OS. e Western blotting analysis of lysates from U2OS UBXN7-KO clone #1 transfected with the indicated plasmids and treated or not with MG132. Quantification and statistical analysis as in b. Western blot quantifications values obtained for each experiments are shown in Additional file 3: Table S1