Fig. 3: S367p-induced destabilization and inactivation of p53.

A H1299 cells were transfected with VprBP and/or p53 expression plasmids for 48 h in the presence or absence of B32B3 (3 µM) as indicated on the top. Extracts were prepared in lysis buffer and Western blot analysis was performed with antibodies against p53, p53S367p, FLAG, and VprBP. B Whole cell lysates from (A) were subjected to immunoprecipitation using p53 DO-1 antibody. The binding of VprBP to p53 was analyzed by Western blotting. C Total RNA was isolated from the H1299 cells transfected with VprBP and/or p53 expression plasmids as in (A), and subjected to RT-qPCR analysis with p21 and BTG2 specific primers listed in Supplementary Table S1. Data represent the means ± SD of three independent experiments. P values were calculated using two-way ANOVA with post-hoc Tukey’s test for multiple comparisons. ***P < 0.001 versus p53; ^###P < 0.001 versus p53 + FLAG-VprBP. (See also Supplementary Fig. S8A). D ChIP assays were performed in the H1299 cells transfected with VprBP and/or p53 expression plasmids as in (A) using p53 DO-1 antibody. Precipitated DNA was amplified with specific primers for p21 and BTG2 listed in Supplementary Table S2. Data are represented as mean ± SD of three independent experiments. P values were calculated using two-way ANOVA with post-hoc Tukey’s test for multiple comparisons. ***P < 0.001 versus p53; ^###P < 0.001 versus p53 + FLAG-VprBP. (See also Supplementary Fig. S8B).