Actin polymerization affects the Ser315 phosphorylation and ubiquitination of p53.

<p><i>A.</i> U2OS cells transfected with HA-actin were treated with ETO (10 µM) or untreated as control for 12 h. The cells were then harvested and whole cell proteins were extracted for western blottings with indicated antibodies. <i>B.</i> Western blottings were analyzed using Image J software. Results are presented as mean ± SD of values from three independent experiments. <i>C.</i> U2OS cells transfected with HA-actin. At 24 h after the transfection, cells were treated with or without ETO for 12 h, followed by the treatment of 20 µM MG132 for 6 h. Whole cell extracts were immunoprecipitated with anti-p53 (DO-1) antibody and analyzed by western blotting with anti-p53 (FL-393) antibody. <i>D</i>. Cells were transfected with Aurora kinase A siRNA, and 24 h after transfection, cells were treated with ETO (10 µM) or untreated as control for 12 h. Then, cells were harvested and whole cell proteins were extracted for western blotting (a). Western blottings were analyzed using Image J software. Results are presented as mean ± SD of values from three independent experiments (b). <i>E</i>. Cells were harvested and subjected to immunoprecipitations using anti-Aurora A antibody and analyzed by western blotting. <i>F</i>. Cells were transfected with actin or actin siRNA, or treated with ETO (10 µM, 12 hours) or untreated as control, or CD (0.01µg/ml) and Jas (50 nM) for 2 h. Cells were then harvested and subjected to immunoprecipitations using anti-p53 antibody and analyzed by western blotting (a). Western blotting results were analyzed using Image J software. Results are presented as mean ± SD of values from three independent experiments (b). All Statistical differences were determined by One-way ANOVA. **, P<0.01.