Figure 6.

Phosphorylation of the wild-type and mutant Cdt1 proteins by various kinases in vitro. (A) Standard reaction mixtures for in vitro phosphorylation by Cdk2/Cyclin A and Cdc2/Cyclin B kinase complex were incubated with wild-type and mutant Cdt1 proteins at the same concentration for 1 hr at 37°C, and products were analyzed on 5–20% SDS-PAGE. The gel was stained with silver (left panel) and dried for autoradiogram (right panel). (B) In vitro phosphorylation assays were conducted with Cdc7/Dbf4 kinase (Cdc7 WT) and Cdc7 kinase dead mutant (Cdc7 KD). The reaction products were analyzed on 5–20% SDS-PAGE, which was silver-stained (left panel), dried, and autoradiographed (right panel). (C) The levels of Cdt1 phosphorylation in the autoradiograms in A and B were quantified by Fuji image analyzer, expressed as relative values (the maximum level of kinase activities taken as 100) and were plotted.