In vitro ATR kinase assay

In vitro ATR kinase assay was perforem by Eurofins Pharma Discovery Services (Dundee, UK). Briefly, ATR/ATRIP (human) is incubated in assay buffer containing 50 nM GST-cMyc-p53 and various concentrations of compounds. The reaction is initiated by the addition of the Mg/ATP (10 μM) mix. After incubation for 30 minutes at room temperature, the reaction is stopped by the addition of stop solution containing EDTA. Finally, detection buffer is added, which contains d2-labelled anti-GST monoclonal antibody, and a Europium-labelled anti-phospho Ser15 antibody against phosphorylated p53. The plate is then read in time-resolved fluorescence mode and the homogeneous time-resolved fluorescence (HTRF) signal is determined according to the formula HTRF = 10000 × (Em_665nm/Em_620nm). Results shown represent average of three independent experiments.