FIGURE 3.

Coexpression of GAS41 and PP2Cβ affects the phosphorylation status and stability of p53 in cells. A, specific reduction in phosphorylation at serine 366 on p53 in cells. FLAG-PP2Cβ1 and HA-GAS41 were ectopically expressed in U2OS cells for 24 h followed by UV irradiation (lanes 2–5). Whole cell extracts were analyzed by immunoblotting with the indicated antibodies. Representative results from one of the three independent experiments are shown. B, time course of p53 phosphorylation at serine 366. U2OS cells were transfected with control and PP2Cβ siRNAs followed by UV irradiation after 24 h of transfection. Whole cell extracts were analyzed by immunoblotting with the indicated antibodies. The p53 levels are normalized against β-actin bands, and the relative levels are shown in the p53 immunoblot panel. C, effects of GAS41-PP2Cβ1 complex in U2OS cells to UV-induced cytotoxicity. To assess UV sensitivity upon GAS41-PP2Cβ1 overexpression, GAS41 and PP2Cβ1 were ectopically overexpressed individually or together in U2OS cells, and UV-mediated cytotoxicity was evaluated using a Promega CellTiter 96 AQueous One solution and a fluorescence plate reader. The value obtained at 490 nm fluorescence reading after UV irradiation is divided by the value obtained from non-stressed cells, and the average values of triplicate experiments are shown in the graph. ▵, vector control; ◊, GAS41; ▴, PP2Cβ; ■, GAS41+PP2Cβ. Error bars indicate S.D.