Fig. 5

USP5 interacts with c-Jun and promotes c-Jun protein stability. A Immunofluorescence images of HA-USP5 (red) and FLAG-c-Jun (green) in HEK293T cells. DAPI was used as a nuclear stain (blue). B Immunoprecipitation experiments showed the endogenous interaction between USP5 and c-Jun in T24 cells. C Cells were transfected with increasing amounts of the HA-USP5 (0, 100, 200, 500 ng) and FLAG-c-Jun plasmids, and western blotting was performed to determine the effect of USP5 protein levels on c-Jun expression in HEK293T cells. D Western blot analysis of c-Jun expression in USP5-deficient T24 cells. E HEK293T cells were transfected with FLAG-c-Jun with or without HA-USP5 as indicated. Western blot analysis of c-Jun stability after treatment with CHX (50 µg/mL) for the indicated time. GAPDH served as a control. F HEK293T cells were transfected with FLAG-c-Jun with or without HA-USP5 C335A as indicated. Western blot analysis of c-Jun stability after treatment with CHX (50 µg/mL) for the indicated times. GAPDH served as a control. G Western blot analysis of c-Jun stability in USP5-deficient cells after treatment with CHX (50 µg/mL) for the indicated time. GAPDH served as a control. H HEK293T cells were cotransfected with FLAG-c-Jun and Myc-Ub with or without HA-USP5 or HA-USP5 C335A, treated with MG132 (10 µM) for 6 h and then subjected to ubiquitination assays. The data are representative of 3 independent experiments. Statistical significance was analysed by ANOVA or Student’s t test. **P < 0.01