Fig. 5

USP3 stabilizes SUZ12. a Flag-tagged USP3 plasmid was transfected into GC cells. Immunoprecipitation was performed with anti-flag antibody, and pre-immune normal mouse immunoglobulin G (nm IgG) was used as a control. Western blot analysis was performed with an anti-Myc (SUZ12) antibody. b Cell lysates of GC cells were immmunoprecipitated by an anti-USP3 antibody or the control antibody, normal rabbit immunoglobulin G (nr IgG). Western blotting was performed with an anti-SUZ12 antibody. All experiments were repeated 3 times, and similar findings were obtained. c Flag-USP3 was transfected into BCG-823 cells. After treating the cells with cycloheximide (CHX; 50 mg/mL) for the indicated time intervals, expression of SUZ12 and USP3 was examined by western blot (up) using the indicated antibodies. The intensity of endogenous SUZ12 expression for each time point was quantified by densitometry (down). d USP3 or vector was transfected into BCG-823 cells for 48 h. After the cells were treated with or without 10 mmol/L MG132 for 6 h, SUZ12 was immunoprecipitated with anti-SUZ12 antibody, and the polyubiquitination of SUZ12 was examined by Western blot using anti-ubiquitin. IP: immunoprecipitation; Wb: Western blot. e The scr or USP3 siRNA was transfected into BCG-823 cells for 48 h and then cells were treated with or without 10 mmol/L MG132 for 6 h. Extracts were immunoprecipitated with an anti-SUZ12 antibody, and the polyubiquitination of SUZ12 was examined by Western blot using anti-ubiquitin. The experiments were repeated three times, and representative images of the blots are shown