FIGURE 6.

Repression of Cdc25A or Cdc25B by shRNA impairs Cdk1-cyclin B complex assembly. A, HeLa cells were transfected with plasmids, encoding shRNA targeting Cdc25A or firefly luciferase as a control and, 24 h after transfection, were synchronized by a double thymidine block. After release, cells were collected at different time intervals and used for FACS to confirm synchronization and trace stages of the cell cycle (not shown) and for preparation of lysates. Left, cyclin B was immunoprecipitated from 500 μg of lysates using the antibodies indicated, and proteins were resolved on SDS-PAGE and analyzed by WB. Density of Cdk1 bands was measured using ImageJ software, and their relative intensities were calculated as the ratio to the density of the Cdk1 band at 9 h after release (mitosis), considered as 1. Results are indicated below the Cdk1 blots. Right, 50 μg of lysates used for IP analyzed by WB to monitor repression of Cdc25A and evenness of loading. B, similar to A, but plasmids expressing shRNA against Cdc25B phosphatase were used. C, similar to A and B, but both Cdc25A and Cdc25B were repressed using corresponding shRNA-expressing constructs. D, data of relative density measurements at time point 9 h were used for statistical analysis in three independent experiments. Error bars, S.D.