In vitro phosphorylation of p53-Ser15 by immunoprecipitated ATM.

The catalytic activity of ATM protein immunoprecipitated with anti-FLAG antibody from 293 cells transiently transfected with pcDNA3-YZ5 or pcDNA3-YZ5/8921T was assayed in vitro against the GST-p53 (amino acids 1-100) substrate. Kinase reactions were Western blotted and tested with an anti-p53–Ser15 phosphospecific antibody, or with an anti-ATM antibody, to verify the amount of immunoprecipitated ATM protein per lane. Equal amounts of GST-p53 were loaded. Representative data from 3 independent experiments are shown (left panel). Phosphorylation of recombinant GST-p53 (amino acids 1-100) by pcDNA3-YZ5/8921T was standardized for kinase activity of pcDNA3-YZ5 (= 1), and was shown as fold increase. Data were expressed as the mean (± SE) of 3 independent experiments (right panel).