FIGURE 5.

Effect of cis/trans conformation of ATR on its kinase activity. (A) In vitro phosphorylation of purified GST-p53 was carried out to assess the checkpoint kinase activity of IPed ATR from the cytoplasmic and nuclear fractions of ATR^flox/− cells expressing co-transfected FLAG-ATR WT, mutants P429A, or S428A plus Myc-ATRIP constructs. GST-p53 phosphorylation was monitored by Western blot using phospho-S15 p53 antibody. (B) Phosphorylation of ATR on T1989 was monitored to confirm the checkpoint kinase activity of ATR WT, S428A, and P429A mutant proteins in ATR^flox/− cells after transfection with the respective plasmid construct. The nuclear fraction was collected from UV+/− irradiated cells after 2 h of recovery.