ATM-dependent p53 phosphorylation after DNA damage. (A) Western blot detection of Ser15-phosphorylated p53, Ser20-phosphorylated p53, total p53, and α-tubulin (as a control for the amount of cell lysate loaded) in lysates of immortalized lymphoblastic cells from healthy subjects (LCL-wt1 and LCL-wt2), patients with HD (LCL-HD1 and LCL-HD2), and a patient with AT (AT65RM), before (–) and 30 minutes after (+) X-irradiation (IR; 5 Gy) of the cells in culture. Representative data from 3 independent experiments are shown. (B) Western blot detection of GST-p53 phospho-Ser15 in an in vitro phosphorylation assay with immunoprecipitated ATM from AT cells (AT65RM), wild-type cells (LCL-wt1), and HD cells (LCL-HD1 and LCL-HD2); equivalence of loading of GST-p53 is shown by Coomassie brilliant blue staining (third panel). Extents of phosphorylation of recombinant GST-p53 (1-100) are shown relative to that of LCL-wt1 (= 1) (bottom graph). Data represent mean values from 3 independent experiments. Equal amounts of ATM protein (top panel) were immunoprecipitated from LCL-wt1, LCL-HD1, and LCL-HD2; no ATM protein was immunoprecipitated from AT65RM. (C) Whole lysates of cells from a healthy subject (LCL-wt2), patients HD1 and HD2 (LCL-HD1 and LCL-HD2), and a patient with AT (AT65RM) were immunoprecipitated with anti–Chk2 antibody before (–) and 1 hour after (+) X-irradiation (5 Gy). Immunoprecipitated Chk2 protein (detected by Western blotting with anti–Chk2 antibody; top panel) was used in an in vitro kinase assay with recombinant GST-Cdc25C as the substrate (detected with Coomassie brilliant blue; third panel), phosphorylation of which is shown by ³²P incorporation (by autoradiography; second panel). Extents of phosphorylation of recombinant GST-Cdc25C (167-267) after X-irradiation are shown relative to that before X-irradiation (= 1) (bottom graph). Data represent mean values from 3 independent experiments.