Figure 1.

USP15 binds to and stabilizes p53. (a) Schematic representation of USP15 and its deletions. (b) The C-terminal domain of USP15 binds to p53 in GST pull-down assays. An aliquot (0.015 μg) of GST, GST-USP15(1–953), GST-USP15(1–547) or GST-USP15(548–953) was incubated with His₆-p53 (0.3 μg) in 0.7 ml of buffer, followed by precipitation using Glutathione-Sepharose beads. All precipitated components were analyzed by western blotting and probed for GST and His₆. (c) The endogenous USP15 co-immunoprecipitated with endogenous p53 from crude lysates of HEK293 cells. Crude proteins (2000 μg) extracted from HEK293 cells were incubated with the anti-p53 antibody (2 μl) that pre-immobilized on protein A Sepharose (30 μl). After centrifugation, all components in the pellet were analyzed by western blotting using anti-USP15 antibody. (d) USP15 promoting p53 stability is dependent on its enzymatic activity. Flag-USP15 or Flag-USP15 C269S was overexpressed in HEK293 cells for 24 h. Cells lysates were prepared and probed for p53, p21, actin and Flag. Actin was used for the loading control. (e) The wild-type USP15 or mutant USP15 binds to p53 in GST pull-down assays. An aliquot (0.015 μg) of GST, GST-USP15(WT) or GST-USP15(c269s) was incubated with His₆-p53 (0.3 μg) in 0.7 ml of buffer, followed by precipitation using Glutathione-Sepharose beads. All precipitated components were analyzed by western blotting and probed for GST and His₆. (f) Overexpression of USP15 increases the levels of p53 in p53-knockdowned cells. HEK293 cells with p53 knockdown by shRNA were transfected with the plasmid harboring USP15 cDNA for 24 h. Crude protein extracts were prepared and probed for p53, Flag and actin.