v-cyclin-associated kinase activity does not require p27^KIP1. (A) Lysate from BC-3 cells was immunodepleted for p27^KIP1 by 3 consecutive rounds of immunoprecipitation with anti-p27^KIP1 antibody. In the control, lysate was immunoprecipitated with rabbit IgG. Subsequently, the depleted lysate was subjected to immunoprecipitation by anti-v-cyclin antibody and in vitro kinase assay toward GST-Rb and Histone H1. Kinase activity was determined by autoradiography after SDS-PAGE (12%). The p27^KIP1depleted v-cyclin immunoprecipitate and lysate (40 μg) was resolved by SDS-PAGE (12%) and immunoblotted with antibody against p27^KIP1. (B) U2OS cells were transfected with Myc-v-cyclin, siRNA against p27^KIP1, and unrelated siRNA (Lkb1) as a control. Cells were lysed 48 hours after transfection and lysates were immunoprecipitated with anti-Myc antibody. Kinase activity was determined by in vitro kinase assay toward GST-Rb and Histone H1 and detected by autoradiography after SDS-PAGE (12%). Total protein levels of p27^KIP1, v-cyclin, and actin (loading control) were determined by immunoblotting. — indicates no siRNA.