Figure 8.

Restoration of p27^kip1 kinase activity with recombinant p27^kip1. Dermal fibroblasts were isolated from newborn mice with a kip^−/− phenotype (Koff et al., 1996). Density-arrested cultures were stimulated with fresh medium containing 10% CS serum and 10 ng/ml PDGF for 20 h. Cell extracts containing 40 μg of protein were subjected to immunodepletion with p27^kip1, cyclin D3, p27^kip1 plus cyclin D3 antibodies, or used without any further treatment. Immunodepleted cell extracts were incubated in the presence or absence of recombinant kip protein for 30 min at 30°C and immunoprecipitated with p27^kip1 antibody-linked beads. Immunoprecipitates were analyzed for in vitro kinase activity using GST-Rb as a substrate. Phosphorylated proteins were separated by SDS-PAGE and visualized by autoradiography.