p27^KIP1 is phosphorylated by v-cyclin-CDK6 in vivo. (A) Lysates from BC-3 cells were separated by gel filtration chromatography. In the left panels the peak fraction for p27^KIP1 phosphorylation (the 110-kDa fraction from Figure 2A) was immunodepleted of p27^KIP1 with 3 consecutive rounds of immunoprecipitation using anti-p27^KIP1 antibody (depleted +) or with rabbit immunoglobulin G for control (-). The p27^KIP1-and control-depleted extracts were immunoprecipitated with anti-v-cyclin antibody and subjected to an in vitro kinase assay (top left panel). A parallel sample of the same fraction was also immunoprecipitated with anti-v-cyclin antibody neutralized by pretreatment with GST-v-cyclin (block +) or with anti-v-cyclin antibody treated with a nonspecific control protein BSA (-), and assayed for kinase activity toward coprecipitated endogenous p27^KIP1 (top right panel). Kinase activity was determined by autoradiography after 12% SDS-PAGE (P³²-p27KIP1; Kinase). Immunoprecipitated proteins were detected by immunoblotting with anti-v-cyclin and anti-p27^KIP1 antibodies (WB). The p27^KIP1 depleted total lysate was immunoblotted with antibodies against p27^KIP1 and v-cyclin (left, 2 bottom panels). (B) Total extracts from BC-3 cell line were immunodepleted for CDK2, CDK4, and CDK6 by 3 consecutive rounds of immunoprecipitation with anti-CDK2, anti-CDK4, or anti-CDK6 antibodies, respectively. In the control (contr.), lysates were subjected to immunoprecipitation with rabbit IgG. The depleted extracts were subjected to immunoprecipitation by anti-v-cyclin antibody and in vitro kinase assay toward GST-p27^KIP1. Kinase activity was determined as in panel A (top panel). Successful depletion of individual proteins from the total lysates was confirmed by immunoblotting with antibodies against CDK2, CDK4, and CDK6 (WB).