FIG. 6.
Inhibition of cyclin D3-cdk4 activity by p27kip1 in mixed cell extracts. Extracts of density-arrested BALB/c 3T3 cells were boiled for 5 min and clarified by centrifugation (designated “G0 extracts”). G0 extracts were mixed at the indicated ratios with extracts (200 μg) of proliferating p27/p21−/− cells and incubated at 30°C for 30 min. Mixed cell extracts were immunoprecipitated with antibody to cyclin D3 (A and B) or cyclin A (C and D). Immune complexes were assayed for cyclin D3-associated activity (A) or cyclin A-associated activity (C) or were immunoblotted with antibody to p27kip1 (B and D). Based on comparisons with known amounts of recombinant p27kip1, 100 μg of G0 extract contains approximately 7 ng of p27kip1. Ab, antibody. IP, immunoprecipitation.