FIG. 1.
Lack of effect of p27kip1 immunodepletion on cyclin D3-associated kinase activity in BALB/c 3T3 cells. Density-arrested BALB/c 3T3 cells were mitogenically stimulated with 10 ng of platelet-derived growth factor per ml and 10% serum and harvested at the indicated times. (A) Cell lysates (40 μg) were immunoblotted with antibody to cyclin D3. (B and C) Cell lysates (80 μg) were incubated with antibody to p27kip1. Immune complexes were pelleted with protein A-agarose beads and immunoblotted with antibody to cyclin D3 (C). The p27kip1-depleted supernatant was immunoprecipitated and immunoblotted with cyclin D3 antibody (B). (D) Cell lysates (40 μg) were immunoblotted with antibody to p27kip1. (E and F) Cell lysates were immunoprecipitated with preimmune serum (E) or antibody to p27kip1 (F). Immune complexes were removed by centrifugation with protein A-agarose beads, and supernatants were immunoprecipitated with cyclin D3 antibody. Immunoprecipitated material was assayed for cyclin D3-associated kinase activity using GST-Rb as substrate.