v-cyclin-CDK6 phosphorylates p27^KIP1 predominantly on Ser10. BC-3 (A) and BC-1 (B) whole-cell extracts were immunoprecipitated with anti-v-cyclin antibody and assayed for kinase activity toward GST-p27wt (WT), GST-p27S10A (S10A), GST-p27T187A (T187A), and GST-p27S10AT187A (S10AT187A). The kinase reactions were resolved by SDS-PAGE (12%) followed by autoradiography (P³²-p27KIP1; Kinase). Immunoprecipitation of equal levels of v-cyclin was confirmed by immunoblotting with anti-v-cyclin antibody (WB). The extent of [³²P] incorporation into the wild-type and mutants of GST-p27^KIP1 in BC-3 (C) and BC-1 (D) was quantified from the autoradiographs by Typhoon 9400 Image Quant analyzer as detailed in “Materials and methods.” The level of [³²P] incorporation by wild-type GST-p27^KIP1 was defined as 100%. Data present the average of 3 independent experiments.