FIG. 2.
Inhibition of CDK4 and CDK2 by induction of p16INK4a. (A) EH1 cells were grown for 48 h with or without IPTG and CDK4 immunoprecipitates, prepared with or without competing peptide antigen, were tested for the ability to phosphorylate the carboxy-terminal domain of pRb in vitro. (B) Thirty-microgram aliquots of lysates from EH1 cells grown in the presence or absence of IPTG were immunoblotted with an antibody specific for pRb phosphorylated at serine 780 (31). (C) Lysates from IPTG-treated and untreated cells were immunoprecipitated with a control nonimmune serum (immunoglobulin G [IgG]) or with antisera against CDK2, cyclin E, or cyclin A as indicated. The immune complexes were then tested for kinase activity using the carboxy-terminal domain of pRb as the substrate. The results were quantitated by phosphorimaging and expressed as the percentage reduction in kinase activity after induction of p16INK4a. The values presented are from two independent experiments.