Figure 3.

USP15 deubiquitinates p53. (a) USP15 overexpression deubiquitinates p53. HEK293 cells were co-transfected with the indicated constructs. Twenty-four hours after transfection, cells were treated with MG132 (20 μg/ml) for 5 h. Cell lysates were prepared and precipitated by Ni²⁺-Sepharose beads and probed for p53, GFP and Flag. (b) USP15 antagonizes the destabilization of p53 by MDM2. HEK293 cells were co-transfected with the indicated constructs. Twenty-four hours after transfection, cell lysates were prepared and antibodies were used to probe for p53, GFP, Flag and actin. (c) Quantification of b. The results are shown as a percentage of the p53/actin ratios with the control, and are averaged from three independent experiments (means±s.d.). (d) Regulation of subcellular localization of p53 by USP15. HEK293 cells transfected with the indicated constructs were treated with MG132 (10 μM). After 16 h, cells were harvested and fractionated as described in 'Materials and methods' section. Cellular fractions were analyzed by western blotting using the indicated antibodies. A cytoplasmic marker protein, tubulin and a nuclear markers protein, lamin A/C, were used as controls to confirm the quality of fractions. Abbreviations: C, cytoplasmic fractions; N, nuclear fractions.