Fig. 4: Nuclear p53 stability and activity modulated by S367p and acetylation.

A Control, VprBP-depleted, VprBP/VprBPK194R-rescued U2OS cells were treated with etoposide (40 µM) in the presence or absence of B32B3 (0.5 µM) for 24 h. Cytoplasmic extracts, and nuclear extracts were prepared and analyzed by Western blotting with antibodies against p53, p53S367p and p53ac. B H1299 cells were transfected with FLAG-VprBP and/or p53, p53S367A or p536KQ for 48 h in the presence or absence of B32B3 (3 µM). Cytoplasmic extracts and nuclear extracts were prepared and analyzed by Western blotting with indicated antibodies. C Total RNA was isolated from the H1299 cells transfected with VprBP/p53 expression plasmids as in (B), 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. S10A). D ChIP assays were performed in the H1299 cells transfected with VprBP/p53 expression plasmids as in (B) 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. S10B).