ATM-dependent c-Abl activation in vitro. Whole lysates of cells from a healthy subject (LCL-wt2), patient HD4 (LCL-HD4), and a patient with AT (AT65RM) were immunoprecipitated with anti–c-Abl antibody before (–) and 1 hour after (+) X-irradiation (10 Gy). Immunoprecipitated c-Abl protein (detected by Western blotting with anti–c-Abl antibody; top panel) was used in an in vitro kinase assay with recombinant GST-CTD 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-CTD after X-irradiation are shown relative to that before X-irradiation (= 1) (bottom graph). Data represent mean values from 3 independent experiments.