Fig. 3

USP7 sustains the protein stability of PLK1 as a deubiquitinase. a DU145 and VCaP cells were transfected with scrambled or USP7 siRNAs. Protein levels of USP7, PLK1 and β-actin were assessed by immunoblotting. b DU145 cells were transfected with increasing amounts of pCMV-USP7-Flag as indicated. The PLK1 protein level was assessed by immunoblotting. c DU145 was transfected with scrambled or USP7 siRNAs. Cells were treated with 50 μg/mL cycloheximide (CHX) for the indicated times, and endogenous PLK1 protein levels were monitored over time by immunoblotting. PLK1 protein was quantified by greyscale analysis. d DU145 was transfected with scrambled or USP7 siRNAs. Cells were treated with MG132 for 8 h, and PLK1 or USP7 protein was detected by immunoblotting. PLK1 protein was quantified by greyscale analysis. Standard deviation represents three independent experiments (Additional file 4: Figure S4). e and f Impact of USP7 on PLK1 ubiquitylation in vitro. HEK293T cells were transfected with increasing amounts of pCMV-USP7-Flag or pCMV-USP7(C223S)-Flag together with pCDNA3.1-Ubi-HA and pCMV-PLK1, and then were treated with 25 μM MG132 for 8 h. Immunoprecipitation was performed with anti-PLK1 antibody, and subjected to western blot with an ubiquitin or PLK1 antibody