In vitro phosphorylation of p53 Ser15 by N1650S ATM kinase. FLAG-tagged ATM protein was immunoprecipitated with anti–FLAG antibody from 293 cells transiently transfected with wild-type ATM expression vector pEBS-YZ5, variant ATM expression vector pEBS-YZ5/4949G, or mock transfectant pEBS7. Equal amounts of FLAG-tagged wild-type and variant ATM (detected with anti–FLAG antibody) were immunoprecipitated (top panel). Recombinant GST-p53 (1-100) was subjected to an in vitro phosphorylation assay using the immunoprecipitated, FLAG-tagged wild-type and variant ATM, and GST-p53 phosphorylated at Ser15 was detected by using anti-p53 Ser15 phosphospecific antibody (second panel). Equal amounts of substrate (GST-p53) were present, as shown by Coomassie brilliant blue staining (third panel). The extent of phosphorylation of recombinant GST-p53 (1-100) is shown relative to that of pEBS-YZ5 transfectant (= 1) (bottom graph). Data represent mean values from 3 independent experiments.