Fig. 2. CDC25B is phosphorylated on S230 in vivo. (A) U2OS cells conditionally expressing HA-CDC25B were synchronized by double thymidine block. At the indicated times after release, cells were harvested and subjected to western blot analyses with monoclonal anti-HA and anti-CDC25B-S230-P antibodies. Actin was used as loading control. The cell-cycle distributions were determined at each time point by flow cytometry analyses and shown as the percentage of cells in G1-, S- or G2-phase. (B) HeLa cells transiently expressing the DsRed1-tagged DNA ligase I (red) were immunostained with anti-CDC25B-S230-P (S230p, green) and anti--tubulin (blue) antibodies. (C) HeLa cells were immunostained with anti-CDC25B-S230-P (green) and anti--tubulin (red) antibodies and co-stained with DAPI. Cells were selected in interphase or at different stages of mitosis. Yellow regions in merged images indicate centrosomal colocalization of the two proteins. (D) U2OS cells expressing HA-tagged CDC25B were immunostained as in C but, in comparison to control cells, labelling with the anti-CDC25B-S230-P antibody in competition with CHK1-phosphorylated recombinant HA-CDC25B (CDC25B-p) or unphosphorylated (CDC25B) protein. (E) HeLa cells were transfected with control or CDC25B siRNA. Cells were harvested 72 hours later and immunostained with the anti-CDC25B-S230-P (red) and anti--tubulin (green) antibodies (left panel) and the percentages of cells with centrosomes stained by the anti-CDC25B-S230-P antibody were determined (right panel). Magnification, 1000x.