Cyclin D3/cdk4 is the kinase that is most able to sequester the inhibitory activity of p27^kip1 but not p21^cip1 and p57^kip2. Increasing amounts of GST-p27^kip1, GST-p21^cip1, or GST-p57^kip2 were used to inhibit the kinase activity of cyclin E/cdk2 in vitro (A, left panel). The right-hand of panel A shows the expression levels of cyclin D1, D2, D3, cdk4, and cdk6 in individual D-type cyclin/cdk4 or cdk6 complexes (cyclin D1, cyclin D2, and cyclin D3 are labeled as D1, D2, and D3, whereas cdk4 and cdk6 were labeled as K4 and K6, respectively). The expression levels of D-type cyclins and cdk4 or cdk6 were detected by 9E10 antibody and anti-cdk4 or anti-cdk6 antibodies, respectively. The kinase activity of various D-type cyclin/cdk's was measured in vitro using GST-Rb as a substrate (B, left). Cyclin E/cdk2 was used as a positive control. The ability of GST-p27^kip1, GST-p21^cip1, and GST-p57^kip2 but not GST to inhibit the kinase activity of cyclinE/cdk2 is shown in panel C, left (lanes 1-3). The left subpanel of C shows the ability of various D-type cyclin/cdk's to sequester the inhibitory activity of p27^kip1, p21^cip1, and p57^kip2 on cyclin E/cdk2 kinase in vitro. The amounts of different combinations of D-type cyclin/cdk used in this experiment were the same as those shown in panel B, and they were all derived from the same extracts. The amounts of individual D-type cyclin/cdk4 or cdk6 lysates, GST-p27^kip1, GST-p21^cip1, and GST-p57^kip2 used in the kinase assay are also indicated in the right-hand subpanels of B and C. The bar graphs in panel D show quantitatively how efficient each combination of D-type cyclin/cdk is in sequestering the inhibitory activity of p27^kip1, p21^cip1, and p57^kip2 on cyclin E/cdk2 kinase. The cyclin E/cdk2 kinase activity detected in the presence of p27^kip1 or p21^cip1 or p57^kip2 (left graph, lane 3 for all blots in C; right graph, lane 3 for all blots in C) was used to set the baseline and was valued at 1.