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. 2014 Nov 19;290(1):556–567. doi: 10.1074/jbc.M114.614701

FIGURE 6.

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.