Recruitment of Chk2^Cds1 to T275S278-phosphorylated Sid4 compensates for the SPB activation defect of cut12.1. (A and F) Representative plots of monopolar spindle counts as for Fig. 1 C. See also Fig. S3 A. In each case, n = 3. (B) Yeast two-hybrid comparisons of the indicated constructs. For full dilution series, see Fig. S3 B. Mrc1 is a validated Chk2^Cds1 partner (Tanaka and Russell, 2001). SD-L-T, SD-Leu-Trp; SD-L-T-H, SD-Leu-Trp-His. (C and D) BiFc assays of fluorescence generated between Chk2^Cds1.nYFP and Sid4.cYFP in the indicated strains. Each field shows cells from cultures of different strains as indicated. Cell walls of the strain named in red having been stained by transient suspension in red fluorescent lectin to identify it in the mixed field. The images are maximum projections of deconvoluted z stacks that span the diameter of the cell. See also Fig. S3 C. n = 3. Red arrows indicate signals at SPBs of wild-type sid4⁺ cells, and white arrows indicate signals at SPBs of sid4.T275A278AcYFP cells. BF, brightfield. (E) GFP-Trap precipitates from the indicated Chk2^Cds1.GFP dma1.Δ strains probed with Sid4 antibodies to detect coprecipitating Sid4. Coprecipitation was abolished by simultaneous phospho-blocking mutation at 275 and 278. n = 3. IP, immunoprecipitation. (F) A switch from 20 µM thiamine to the indicated concentrations 24 h before the temperature shift to 36°C de-repressed transcription of the Chk2^Cds1.GFPn in a dose-dependent manner. n = 3.