Fig. 1. CHK1 phosphorylates CDC25B on residues S230 and S563 in vitro. (A) Alignment of corresponding CHK1 phosphorylation sites in human CDC25A and CDC25B. (B) Recombinant HA-CDC25B protein was phosphorylated (+) or not (–) by CHK1. Western blot analyses were performed with anti-CDC25B-S230-P (S230p) or anti-CDC25B-S563-P (S563p) antibodies in the presence (+) or absence (–) of an excess of competing phosphorylated peptides (pp). (C) HeLa cells were either not treated (NT) or transiently transfected with wild-type EYFP-CDC25B (WT), EYFP-CDC25B S230A or EYFP-CDC25B S563A. Cell lysates were analysed by western blotting using the anti-CDC25B-S230-P or anti-CDC25B-S563-P antibodies (S230p or S563p, respectively). The total amount of expressed fusion CDC25 protein was detected with an anti-GFP antibody, and actin was used as loading control.