Figure 4.

In vitro kinase assay to determine the CDK4-inhibitory activities of human P16 wild-type and mutant proteins. Each reaction mixture included 3 units of CDK4-cyclin D2 (∼0.15 μg), 50 ng of GST-Rb790-928, 5 μCi [γ-³²P]ATP, and various amounts of P16 proteins. After reaction at 30°C for 15 min, the reaction mixtures were separated by 10% SDS-PAGE and the incorporation of γ-³²P into GST-Rb protein was quantified using a PhoshoImager (Molecular Dynamics, Inc., Sunnyvale, CA). Filled circles represent hamster P16 wild-type protein, and open circels represent hamster P16 A20P mutant protein. All measurements were performed in triplicate.