Dominant-negative effect on p53-Ser15 phosphorylation in U2OS transfectants.

(A) Western blot analysis of p53-Ser15 phosphorylation after X-IR in clone 1 (left panel) and clone 2 (right panel). Cells were harvested at 0 and 30 minutes after 5-Gy X-IR and analyzed by Western blotting with anti-p53–Ser15 phosphospecific antibody. α-Tubulin was used as a control for protein loading. (B) Fold decrease of p53-Ser15 phosphorylation in clone 1 and clone 2, standardized for the level in irradiated/nonirradiated U2OS-mock (= 1.00). Data were expressed as the mean (± SE) of 2 independent experiments. U2OS cells were transfected with vector alone or pcDNA3-YZ5/8921T. Two independent clones (1 and 2) were isolated and analyzed for expression of wild-type and mutant ATM mRNA by RT-PCR/RFLP analysis using theAlwNI restriction site specific for the 8921T mutation. Clones 1 and 2 expressed wild-type and mutant ATM at a ratio of 1:0.1 and1:0.8, respectively.