FIG. 12.
Dependence of reduced Rb kinase activity on p16INK4A expression after DNA damage. Calu-1 cells infected with pBPSTR1 (C) or pBPSTR1-p16INK4A (clone 14) were untreated (NT) or treated with ADR (A) as described in the legend to Fig. 3. Lysates were subjected to immune precipitation using anti-cdk4 and anti-cdk6 antibodies, and the precipitates were used to phosphorylate GST-Rb in vitro. Kinase assays were analyzed by SDS-PAGE followed by electrophoretic transfer to nitrocellulose. Phosphorylated GST-Rb was detected by exposing the nitrocellulose filter to X-ray film. The presence of equal amounts of cdk4 and cdk6 in the precipitates was then determined by staining the nitrocellulose filter with anti-cdk4 and anti-cdk6 antibodies.