Fig. 3. (A) Cells were synchronized at the G1–S border by a thymidine block for 15 h (lane 1) and subsequent release into complete medium for 3 h (lane 2) or 6 h (lane 3). Cells in M phase were obtained by nocodazole addition for 15 h (50 ng/ml); round mitotic cells were collected (lane 4) and plated in complete medium for 3 and 5 h (lanes 5 and 6). Fifty micrograms of total lysate from each sample were analyzed by immunoblotting with Cdc25 A antibodies. OP, overexpressed Cdc25 A. Cell cycle position was verified by FACS analysis. (B) HeLa cells were synchronized in mitosis with nocodazole and then released into complete medium for 0, 6, 9 and 12 h. Cell cycle position was verified by FACS analysis (Figure 4D). Cdc25 A phosphatase activity was measured as activation of Cdc2–cycB.