Figure 2.

cdc25C activity transiently increases during S-phase. (A). HeLa cells were synchronized before S-phase by double thymidine block. At different times after washing out thymidine, cells were lysed and analyzed by Western blot for the expression of cdc25A (top panel) and cdc25C. Protein extracts were quantitated using Bradford assay (B), and equal amounts of cell extract were loaded for each lane. Western blots show typical examples obtained in four different experiments. Alternatively, HeLa cells were synchronized by double thymidine block or nocodazole and at different times after release were lysed for cdc25C activity measurements or fixed for analysis of BrdU incorporation. cdc25C activity was assessed by immunoprecipitation under nondenaturing conditions. Release of phosphate from 3-OMFP was measured by the associated increase in fluorescence. 3-OMFP phosphatase activity of cdc25C immunoprecipitates isolated at different times after release from double thymidine block. The time points correspond to those analyzed by Western blot in A. (C) The incorporation of BrdU in cells at the same time points. (D) A similar analysis of cdc25C activity in cells released from a nocodazole block and the corresponding data for BrdU incorporation (E). Inset in F: a Western blot analysis of the immunoprecipitate corresponding to the 6-h time point in A and B. Immunoprecipitates made with either C20 or N20 anti-cdc25C antibodies were blotted for the presence of cdc25C (top), cdc25A (middle) and cdc25B (bottom). Histograms are the mean values obtained in three different experiments. Activity is expressed in arbitrary fluorescence units compared against the levels in double thymidine blocked cells, which are essentially the same as those detected in an assay without immunoprecipitate.