Fig. 5

USP16 co-localizes and interacts with c-Myc. a and b Indicated plasmids were transfected into HEK293T cells alone or in combination, and an anti-Flag antibody was used for immunoprecipitation. IP, immunoprecipitation; IB immunoblotting; WCE, whole-cell extract. c and d Exogenous USP16 or c-Myc was immunoprecipitated from PC3 cells using an anti-Flag antibody. Endogenous c-Myc or USP16 was analysed by Western blot. e Interaction between endogenous c-Myc and USP16 in PC3 cells was analysed by Western blot. f Immunofluorescent staining of USP16 and c-Myc in PC3 and DU145 cells. Scale bar, 10 μm. g Flag-c-Myc and HA-ubiquitin were co-transfected with v5-USP16 or v5-C205s, and the ubiquitination of c-Myc was measured using an in vivo ubiquitination assay. Precipitates and WCEs were analysed by Western blot. h Control siRNA (siNC) or siUSP16 was transfected into HEK293T cells. After 48 h, cells were transduced with Flag-c-Myc and HA-Ub for 24 h and then harvested. i HEK293T cells were transfected with v5-USP16, Flag-c-Myc, and different forms of HA-ubiquitins. Cell lysates were immunoprecipitated with an anti-Flag antibody and ubiquitination levels were measured using an anti-HA antibody