Unknown

Kinase reactions consisted of 100 ng of baculovirus expressed GST-CDK1/cyclin B1 complex, 1 µg histone H1 (Boehringer Mannheim, Indianapolis, IN), 0.2 µCi 33P γ-ATP, 25 µM ATP in 50 µL of kinase buffer (50 mM Tris, pH 8.0, 10 mM MgCl2, 1 mM EGTA, 0.5 mM DTT). Reactions were incubated for 45 min at 30 °C and stopped by the addition of cold trichloroacetic acid (TCA) to a final concentration of 15%. TCA precipitates were collected onto GF/C unifilter plates (Packard Instrument Co., Meriden, CT) using a Filtermate universal harvester (Packard Instrument Co.), and the filters were quantitated using a TopCount 96 well liquid scintillation counter (Packard Instrument Co.). Dose response curves were generated to determine the concentration required to inhibit 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in DMSO and evaluated at six concentrations, each in triplicate. The final concentration of DMSO in the assay equaled 2%.