Fig. 2

p53 is less ubiquitinated in the presence of LSH. a, b Regulation of exogenous p53 ubiquitination levels by LSH. H1299 cells were transfected with the indicated constructs and treated with 50 μM MG132 for 4 h. EGFP-p53 was immunoprecipitated with anti-EGFP polyclonal antibodies and immunoblotted with monoclonal anti-p53 (DO-1) antibodies or anti-His antibodies. Densitometry analysis of total ubiquitinated protein content. N = 3, *P < 0.05; **P < 0.01. c Regulation of endogenous p53 ubiquitination levels by LSH. CNE1 and HK1 cells stably expressing vector or LSH were treated with 50 μM MG132 for 4 h, and the cell lysates were immunoprecipitated with anti-p53 polyclonal antibodies and immunoblotted with monoclonal anti-p53 (DO-1) antibodies or anti-Ub antibodies. Densitometry analysis of total ubiquitinated protein content. N = 3, *P < 0.05; **P < 0.01. d Regulation of endogenous p53 ubiquitination levels by LSH. A549 cells stably expressing shControl or LSH shRNA were treated with 50 μM MG132 for 4 h, and cell lysates were immunoprecipitated with anti-p53 polyclonal antibodies and immunoblotted with monoclonal anti-p53 (DO-1) antibody or anti-Ub antibody. Densitometry analysis of total ubiquitinated protein content. N = 3, *P < 0.05; **P < 0.01. e Deubiquitination of p53 in vitro by LSH. Ubiquitinated p53 was incubated with or without purified LSH for 2 h. Reactions were stopped by the addition of SDS-PAGE loading buffer and then blotted with anti-p53 and anti-Ub antibodies (SE, short exposure; LE, long exposure). Densitometry analysis of total ubiquitinated protein content. N = 3, *P < 0.05; **P < 0.01. f LSH cleaves ubiquitinated p53 in vitro. Ubiquitinated p53 was incubated with purified LSH expressed in BL21 cells at 0.0, 0.5, 1, or 2 μg in vitro and then blotted with anti-p53 and anti-Ub antibodies or GST antibody. The protein of purified LSH at was separated by SDS-PAGE. Densitometry analysis of total ubiquitinated protein content. N = 3, *P < 0.05; **P < 0.01. g Deubiquitination of p53 in vitro by LSH truncations. HEK293T cells were transfected with K11, K48, and K63 constructs to form ubiquitin chains, and Ubiquitinated p53 was incubated with or without purified GST-LSH in vitro and then blotted with anti-p53 and anti-Ub antibodies. Densitometry analysis of total ubiquitinated protein content. N = 3, *P < 0.05; **P < 0.01. h Structure of LSH and the three FLAG-LSH constructs used for mapping. i HEK293T cells overexpressing LSH-FLAG or LSH truncations. Endogenous p53, p21, and MDM2 levels were detected using the indicated antibodies. Representative images from three independent experiments are presented. j Deubiquitination of p53 in vitro by LSH truncations. Ubiquitinated p53 was incubated with or without purified LSH truncation mutants in vitro and then blotted with anti-p53 and anti-Ub antibodies. Densitometry analysis of total ubiquitinated protein content. N = 3, *P < 0.05; **P < 0.01. k Determination of the minimal LSH-p53 interaction region. Co-IP assays were performed with an anti-EGFP antibody in HEK293T cells transfected with EGFP-p53 plus one of a series of N-terminal or C-terminal FLAG-LSH mutants. Representative images from three independent experiments are presented