FIGURE 6.
CDK1 activity prevents CDT2 chromatin recruitment. A, HCT116 cells were grown asynchronously or synchronized in prometaphase. Cells were treated with DMSO (none) or the CDK1 inhibitor RO-3306 for 30 min prior to PCNADNA induction by UV irradiation and harvested 90 min later. Endogenous proteins in whole cell extracts and chromatin-bound fractions were analyzed by immunoblotting. B, GST, GST-p21WT, or GST-p21ΔPIP was produced in E. coli and bound to glutathione beads. The protein-coated beads were then incubated with whole cell lysates from asynchronous or mitotic cells as described previously (39). Endogenous PCNA and GST-tagged p21 were detected by immunoblotting. C, HCT116 cells were synchronized in early S phase via double thymidine block and released into fresh medium. Cells were harvested at the indicated time points. Endogenous proteins in whole cell extracts and chromatin-bound fractions were analyzed by immunoblotting.