Fig. 6. Chromosomal cyclin E-cdk2 is active during mitosis. Whole-spindle fractions were tested for kinase activity in the absence (lane 1) and presence of the exogenous substrate pRb-GST (lane 2). Cyclin E was immunoprecipitated from whole-spindle fractions, and the immunoprecipitates were assayed for kinase activity using pRb as the substrate. Minimal kinase activity was associated nonspecifically with protein A-agarose beads (lane 3). However, pRb was strongly phosphorylated by anti-cyclin E immunoprecipitates (lane 4). This activity was blocked by the addition of competing peptide to the antibody incubation (lane 5) or by the addition of 5 µg of the cdk2 inhibitor p27 (lane 6) to the kinase reaction.