Use of ATM immunoprecipitates to determine the effect of PHA on protein kinase activity, using GST-p53 (1-40) as a substrate for ATM kinase. PHA-treated or untreated PBMCs were collected after 72 hours of incubation at 37°C before preparation of lysates. The same amount of total protein was immunoprecipitated with anti-ATM antibody (Ab-3, Oncogene Research). The beads were washed with lysis buffer twice, once with 0.1 mol/L Tris-HCI, pH 7.5 containing 0.5 mol/L LiCl, and finally twice with kinase buffer. Reactions were performed in 25 μL containing 1 μg of GST-p53 (amino acids 1-40), 5 μCi of ATP-γ³²-P in kinase buffer for 30 minutes at 30°C and analyzed for incorporation on SDS-PAGE. − and + refer to without and with PHA in PBMCs from two individuals. Immunoblotting with ATM-5BA antibody was used to detect ATM in the immunoprecipitates.