Supplementary Figure 8: A point mutant of p53 that diminishes PI4,5P₂ binding reduces p53 stability.

(a) 0.1 μM GST-p53 and 0.5 μM untagged HSP27 were incubated with 1 μM PI, PI4P, or PI4,5P₂. GST-p53 was pulled down and the associated HSP27 was analyzed by IB. Representative data of three independent experiments were shown. (b) The indicated p53 constructs were transiently expressed in p53-null H1299 cells for 24 h. p53 was immunoprecipitated and eluted with 0.1 M glycine (pH 2.5) and the associated molecules were analyzed by IB. The graph is shown as mean ± SD of n = 3 independent experiments. (c) In PC3 cells (p53-null), the indicated mutant p53 constructs were stably expressed and analyzed by IB. The graph is shown as mean ± SD of n = 3 independent experiments. (d) The indicated p53 constructs were transfected in p53-null H1299 cells for 24 h. Endogenous HSP27 was immunoprecipitated and the associated p53 was analyzed by IB. The graph is shown as mean ± SD of n = 3 independent experiments. Two-sided paired Student t-tests were used for statistical analysis in b-d. Statistical source data for b-d can be found in Supplementary Table 1. Unprocessed images of the blots in a-d are shown in Supplementary Figure 9.