v-cyclin forms a binary complex with CDK6 and associates with p27^KIP1. Lysates from BC-3 (A) and BC-1 (B) cell lines were separated by gel filtration chromatography and fractions were immunoprecipitated with anti-v-cyclin antibody. Immunocomplexes were then immunoblotted with antibodies against v-cyclin, CDK6, and p27^KIP1 (WB panels). The interactions between v-cyclin and p27^KIP1 were detectable in a short exposure from BC-3 cells, whereas a longer exposure was required for the BC-1 cell line. The immunoprecipitates were subjected to an in vitro kinase assay using recombinant GST-Rb and Histone H1 or coprecipitated endogenous proteins as substrates (Kinase panels). Kinase activity was determined by SDS-PAGE (12%) and autoradiography. (C) BC-3 whole-cell extract was separated as in panel A and the fractions were immunoprecipitated with anti-p27^KIP1 (3 top panels) or anti-CDK2 (3 bottom panels) antibodies. Associated proteins were analyzed in SDS-PAGE by immunoblotting (WB) with anti-v-cyclin, anti-p27^KIP1, anti-CDK6, and anti-CDK2 antibodies as indicated.