FIG. 2.
Lack of effect of p27kip1 immunodepletion on cyclin D3-associated kinase activity in p21−/− MEFs. Confluent p21−/− MEFs were incubated in medium containing 0.1% serum for 30 h and subsequently stimulated with 10 ng of PDGF per ml and 10% serum for 18 h. Cell lysates were immunoprecipitated with preimmune serum (mock depletion, left panel) or antibody to p27kip1 (right panel). Immune complexes were removed by centrifugation with protein A-agarose beads and immunoblotted with monoclonal antibody to cyclin D3 (C). Supernatants were immunoprecipitated with polyclonal antibody to cyclin D3, and immunoprecipitated material was assayed for cyclin D3-associated kinase activity using GST-Rb as substrate (A) or immunoblotted with monoclonal antibody to cyclin D3 (B).