Fig. 3: USP35 deubiquitinates and stabilizes BIRC3.

A and B H460 cells transfected with the USP35 overexpression plasmid, USP35 knockdown plasmid and control plasmid (A) and Anip973 cells transfected with siRNA USP35i-2 and the control siRNA (B) were treated with 50 μg/ml cycloheximide (CHX) for 0, 2, 4 and 6 h. The BIRC3 protein expression level was detected using western blotting. The quantitative analysis of the CHX-chase assay results is shown in the figure. C and D H460 cells transfected with the USP35 overexpression plasmid and control plasmid (C) and Anip973 cells transfected with siRNA USP35i-2 and the control siRNA (D) were treated with or without 10 µM MG132 for 6 h. The BIRC3 protein expression level was detected using western blotting. The quantitative analysis is shown in the figure. E HA-tagged ubiquitin and Flag-BIRC3 were cotransfected into H460 cells in the presence of Myc-USP35 or USP35 knockdown plasmid. Cell lysates were immunoprecipitated with an anti-Flag antibody, followed by immunoblotting with an anti-HA antibody. F K48- or K63-linked polyubiquitin chains and Flag-BIRC3 overexpression plasmid were cotransfected into H460 cells in the presence of wild-type Myc-USP35 or control vector. Cell lysates were immunoprecipitated with an anti-Flag antibody, followed by immunoblotting with an anti-HA antibody. All data are presented as means ± SD. *P < 0.05 and **P < 0.01 based on Student’s t test. All results are representative of three independent experiments.