Fig. 4. Cdc2 is phosphorylated by Wee1 upon Chk1 phosphorylation in orp1-4. (A) orp1-4 chk1-HA cells were synchronised in early G2 by lactose gradient centrifugation and shifted to the restrictive temperature. Total protein extract was prepared and the amount of phosphorylated Cdc2 and Chk1 was investigated by SDS-PAGE and immunoblot analyses against Chk1, total Cdc2 and phosphorylated Cdc2 (Cdc2-YP). (B) Quantification of the blots (A) and comparison with cell cycle parameters. The levels of Cdc2 and Chk1 phosphorylation were corrected for loading. Septation index was determined by aniline blue staining; mitotic index was determined by DAPI staining. (C) Exponentially growing orp1-4, orp1-4 cdc2-1w and orp1-4 cdc2-3w cells were shifted to the restrictive temperature for orp1-4. Cutting was monitored by DAPI staining. (D) Exponentially growing orp1-4, orp1-4 mik1 and orp1-4 wee1-50 cells were shifted to the restrictive temperature for orp1-4. Cutting was monitored by DAPI staining. (E) The orp1-4 mik1-myc strain was shifted to the restrictive temperature for the indicated times. Total protein extract was prepared and the amount of mik1-myc was investigated by SDS-PAGE and western blot analysis. -tubulin was measured as a loading control. (F) The orp1-4 wee1-HA strain was shifted to the restrictive temperature for the indicated times. Total protein was extracted and the amount and phosphorylation state of Wee1 was investigated by SDS-PAGE and western blot analysis.