PCH-2 is required for robust GFP::MAD-2 localization to unattached kinetochores. (A, top) Schematic showing the localization of GFP::MAD-2 to unattached kinetochores after checkpoint activation. Unattached kinetochores are present on the side of the pseudo-metaphase plate lacking a centrosome. Dashed box indicates area shown in images (A, bottom) GFP::MAD-2 signal after zyg-1 RNAi seen in control worms is nearly absent (pch-2) or completely absent (pch-2^†) in pch-2 mutants. Arrows indicate residual GFP::MAD-2 localization. (B) Quantification of kinetochore bound GFP::MAD-2 shows that signal was reduced by a mean of 82% in pch-2 mutants and a mean of 97% in mad-1 mutants. (C) MAD-2 protein levels are unaffected in 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. (D) Quantification of MAD-2 protein level in pch-2 mutants across multiple immunoblots indicates that MAD-2 protein level is 102% of wild type. (E) GFP::MAD-1 properly localizes to unattached kinetochores in pch-2 mutants (F) Quantification of GFP::MAD-1 at unattached kinetochores in wild-type and pch-2 mutants. Error bars in all graphs represent SEM. Bars, 5 µm. **, P < 0.0001.