FIG. 7.
High levels of uncomplexed cyclin D3 in p27/p21−/− MEFs. (A) Boiled and clarified extracts of G0-arrested BALB/c 3T3 cells were mixed with extracts of exponentially growing p27/p21−/− cells at the indicated ratios and incubated for 30 min at 30°C. (Top panel) Mixed cell extracts were immunoprecipitated and immunoblotted with antibody to cyclin D3. (Middle panel) Mixed cell extracts were immunoprecipitated with antibody to p27kip1 and immunoblotted with antibody to cyclin D3. (Bottom panel) Mixed cell extracts were immunodepleted of p27kip1, and depleted extracts were immunoprecipitated and immunoblotted with antibody to cyclin D3. (B) Mixed cell extracts were prepared and incubated as for panel A and were immunodepleted of p27kip1 (+) or mock depleted (−). Depleted extracts were immunoprecipitated (IP) with preimmune serum (PI) or antibody to cdk4 and immunoblotted with antibody to cyclin D3.