FIGURE 1.

Effects of inhibition of Plk in HeLa cells. A, G₂/M arrest in BI2536-treated cells. HeLa cells were released from a double thymidine block. 6 h following release, 100 nm BI2536 or dimethyl sulfoxide (DMSO) as a control were added. Cells were harvested at the indicated times and samples were analyzed by FACS. B, levels of cell cycle proteins in BI2536-treated cells. Cell pellets of the experiment shown in A were lysed in the presence of protease and phosphatase inhibitors as described under “Experimental Procedures” and analyzed by immunoblotting for the specified proteins. C, expression and phosphorylation of cell cycle regulators in nocodazole- or BI2536-treated cells. HeLa cells were released from thymidine block for 11 h into a nocodazole or BI2536 block, as indicated. Asynchronous, S-phase, nocodazole-arrested, or BI2536-arrested cells were harvested, lysed as described above, and analyzed by immunoblotting for the specified proteins. D, Emi1 is phosphorylated in BI2536-treated mitotic cells. 30-μg samples of cell extracts of S-phase cells or BI2536-arrested cells from the experiment shown in C were treated with 10 units/μl of λ phosphatase or buffer alone for 30 min in 30 °C and analyzed by immunoblotting for Cdc27 and Emi1.