Figure 4.

Cyclin A regulates S/G2 phase Cdh1 and Claspin levels.(A) HeLa cells were transfected with ether nonsense (NS), Cyclin A (#1) or Cdh1 siRNA for 24 h then fixed, stained for their DNA content with propidium iodide and sorted into four fractions, G1, early (S1), late S phase (S2), and G2/M phase. The histograms are presented in Figure S5. Fractions were immunoblotted for the indicated proteins. An asynchronous culture is shown as a control (AS). These data are representative of replicate experiments. (B) Thymidine synchronised HeLa cells were harvested at the indicated times after release. Samples were analyzed by FACS for their cell cycle distribution, or lysed and immunoblotted for the indicated proteins. Lysates were also immunoprecipitated for Cyclin A and the immunoprecipitates immunoblotted for the presence of the inactive phosphoTyr15 Cdk2. The level of this inactive form was measured by densitometry and shown on the same graph as the cell cycle distribution (Cdk2 pY15). Mitotic shake-off sample (M) was run as a control. α-Tubulin was used as a loading control.