Mutation of cmt-1 suppresses the checkpoint defect of pch-2 mutants. (A) Mutation of cmt-1 restores checkpoint function in pch-2 mutants. A dashed gray line was drawn at 222 s representing the upper limit of wild-type mitotic timing. 95% of wild-type embryos and 0% of zyg-1RNAi embryos displayed mitotic timing at or below this line. Black lines indicate the mean mitotic timing for each genotype; the whiskers indicate SEM. (B). MAD-2 protein levels are reduced in cmt-1 and cmt-1;pch-2 mutants. Whole worm lysates were first normalized for protein concentration and then serial dilutions were analyzed via immunoblot with an anti–MAD-2 antibody and an anti-α-tubulin antibody serving as a loading control. (C) cmt-1 and cmt-1;pch-2 double mutants show significant reductions in MAD-2 protein levels to 61% and 42% of wild type, respectively, after quantification. (D) cmt-1 and cmt-1;pch-2 mutants show GFP::MAD-2 localization to unattached kinetochores. Bar, 5 µm. (E) Quantification of GFP::MAD-2 signal at unattached kinetochores shows that mutation of cmt-1 partially restores GFP::MAD-2 localization in pch-2 mutants. **, P < 0.0001.
