In Vitro Kinase Assays

Kinase assays were adapted from previous methods.²⁵ Histone H1 protein (2 μg), 50 μM unlabeled ATP, 10 μCi γ-³²P-ATP, and kinase assay buffer containing 1 mM DTT were added to a final volume of 50 μL for in vitro-modified samples and 25 μL for CDK2 immunoprecipitates. The reactions were incubated at RT for 20 min with shaking and stopped with the addition of 2× Laemmli sample buffer with 5% β-mercaptoethanol. Samples were heated at 95 °C for 10 min, cooled, and loaded onto a 4–20% polyacrylamide gel. Following electrophoresis, radioactive histone H1 protein was detected with the Molecular Imager PharosFX System (BioRad, Hercules, CA). Images were quantitated with ImageJ (NIMH, Bethesda, MD). The gel was then stained with Simply Stain (Invitrogen) according to the manufacturer’s protocol.