HSP70 regulates CHIP- and MDM2-dependent degradation of p53 R175H in opposite ways.

<p>Analysis of mutant p53 degradation was performed in double knockout MEF cells (<i>Trp53^−/−, Mdm2^−/−</i>). (<b>A</b>) CHIP-mediated degradation is accelerated by HSP70. (<b>B</b>) HSP70 WT partially inhibits MDM2-dependent p53 R175H degradation. ANOVA statistical test was carried out, where P values were counted for 3 independent experiments. (<b>C)</b> The steady-state level of p53 R175H is increased or decreased upon co-expression of WT or K71S HSP70, respectively. MEF cells were transfected with plasmids encoding p53 R175H and MDM2 together with increasing amounts of WT HSP70. Additionally, for the highest concentration of WT HSP70 titration of HSP70 K71S was applied. 24 hours later the cells were harvested and analysed by immunoblotting with p53 specific antibody (DO-1). The graph depicts the densitometric analysis performed using Image Quant software. Mean and standard deviation (s.d.) of two independent experiments are shown. (<b>D</b>) Overexpression of HSP70 downregulates the level of MDM2-dependent ubiquitination of p53 R175H<b>.</b> Cell based ubiquitination was carried out in MEF cells treated with MG132 (10 µM) for 4 hours. Mutant p53 protein was immunoprecipitated from the lysate using DO-1 antibody and analysed by western blotting using HA tag-specific antibody.