FIG. 4.

Various amounts of p27 differently affect cyclin D3-CDK4 activity in vitro. Cyclin D3-CDK4-HA complexes formed by their coexpression in infected Sf9 cells were incubated in vitro with decreasing amounts of p27-V5 (1, 1/10, 1/20, and 1/30 dilutions) produced in Sf9 cells. This mixture and cyclin D3-CDK4-HA-p27-V5 complexes formed by their coexpression in Sf9 cells were immunoprecipitated (IP) using anti-cyclin D3 or anti-V5 antibodies, assayed for pRb kinase activity, separated by SDS-PAGE, and immunoblotted. Cyclin D3 and the pRb fragment phosphorylated in vitro at Ser780 (P-Rb-780) were detected using specific antibodies; CDK4 and p27 were detected with anti-HA and anti-V5 antibodies.