In vitro kinase assay

In vitro kinase activity was determined by measuring the rate of ADP production in the in vitro kinase reaction using the ADP-Glo Kinase Assay Kit (Promega). Briefly, the EmCDK4/6-EmCyclinD complex was purified by Co-immunoprecipitation as the kinase (Fry et al., 2004) and then incubated with 3 μg of recombination human Rb1 protein (Proteintech) as the substrate in the experiment. The kinase reactions were performed with 1X Kinase Reaction Buffer A [40 mM Tris (pH 7.5), 20 mM MgCl₂, 0.1 mg/mL BSA], 25 μM ATP, 1 mM DTT and incubated at RT, 20 min. The reactions were terminated by ADP-Glo reagent and then detected according to the manufacturer’s instructions. Each data point was collected in duplicate.