v-cyclin-associated kinase activity in PELs requires CDK6. (A) Lysates from PEL cell lines (BC-1, BC-2, BC-3, and BCBL-1) and a KSHV-negative control cell line (JOK-1) were immunoprecipitated with anti-v-cyclin antibody. In vitro kinase activity toward GST-Rb and Histone H1 was determined by autoradiography after SDS-PAGE (12%). The v-cyclin-associated CDK6 and p27^KIP1 were analyzed by immunoblotting the nitrocellulose filter to which kinase reactions had been transferred with anti-CDK6 and anti-p27^KIP1 antibodies. (B) Lysates from BC-3 cells were immunodepleted for CDK2, CDK4, and CDK6 by 3 consecutive rounds of immunoprecipitation with anti-CDK2, anti-CDK4, or anti-CDK6 antibodies. In the control (contr), lysate was immunoprecipitated with rabbit immunoglobulin G (IgG). Subsequently, depleted lysates were 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%). CDK-depleted lysates (40 μg) were resolved by SDS-PAGE (12%) and immunoblotted with antibodies against CDK2, CDK4, and CDK6.