Fig. 3. Chk1, but not Cds1, is required to prevent cutting in orp1-4. (A) Exponentially growing orp1-4, orp1-4 chk1 and orp1-4 cds1 cells were shifted to the restrictive temperature for orp1-4. Cutting was monitored by DAPI staining. (B) orp1-4 cells were shifted to the restrictive temperature for the indicated times and protein extracts were prepared. Cds1 was immunoprecipitated and Cds1 kinase activity was measured using MBP as substrate. Hydroxyurea-treated wild-type cells serve as positive controls. The graph shows quantification of the kinase activity corrected for loading. The immunoprecipitated IgG was used as a loading control. (C) orp1-4 chk1-HA and orp1-4 cdc18-K46 chk1-HA cells were shifted to the restrictive temperature for the indicated times. Total protein was prepared and Chk1 phosphorylation was investigated by SDS-PAGE and western blot analysis.