2.8 In vitro kinase assays

Firstly, enzyme, substrate, ATP and inhibitors AP-7 or the Chk1 inhibitor AZD7762 were diluted in Kinase Buffer, respectively using the CHK1 Kinase Assay Kit (Cat. V1941, Promega Corporation) according to the manufacturer’s protocol. Then, 1 μL of inhibitor or (5% DMSO), 2 μL of enzyme, 2 μL of substrate/ATP mix were added to the wells of 384 low volume plate, and incubated at RT for 1 h. 5 μL of ADP‐Glo™ Reagent in the ADP-GlotTM Assay Kit (Cat. V9101, Promega Corporation) was added to plate and incubated at RT for 40 min. Finally, 10 μL of Kinase Detection Reagent was added and incubate at RT for 30 min, and analyzed by microplate.