Fig. 1. Wee1p and Cdc25p proteins in flp1 mutant S. pombe cells. (A) Wee1p is partially dephosphorylated in a flp1 mutant strain. Cells from asynchronous cultures of wee1_3HA and wee1_3HA flp1 (incubated at 30°C) were processed for western analysis and probed with -Ha monoclonal or -tubulin antibodies. (B) Cdc25p is hyperphosphorylated in the flp1 deletion strain. Cells from asynchronous cultures of wt and flp1 (incubated at 30°C) were processed for western analysis and probed with -Cdc25 polyclonal antibodies. The asterisk marks a reference protein that cross-reacts with the antibody. (C) Cdc25p is hyperphosphorylated in flp1 deletion strains at different stages of the cell cycle. Western blot analysis of Cdc25p protein extracts from cells block in S phase (hydroxyurea, HU, 4 hours), in G2 (cdc2-33, 4 hours at 35.5°C), in mitosis (nda3-KM311, 6 hours at 19°C) or in G1 (cdc10-129, 4 hours at 35.5°C). Blots were probed with -Cdc25 and -Cdc2 polyclonal antibodies, the latter for a loading and proper migration control.