CMT-1 and MAD-2 are required for PCH-2 localization to unattached kinetochores during checkpoint activation. (A) PCH-2 interacts with CMT-1 by yeast two-hybrid assay. PCH-2 is fused to GAL4 DNA-binding domain (bait protein) and CMT-1 is fused to the GAL4-activation domain (prey protein). Empty prey or bait vectors were used as controls in lanes 2 and 3, respectively. (B) PCH-2::GFP-3XFLAG fails to localize to unattached kinetochores in cmt-1 and mad-1 mutant embryos when zyg-1 is knocked down by RNAi. Bar, 5 µm. (C) Quantification of PCH-2::GFP-3XFLAG at unattached kinetochores indicates that mutation of cmt-1 and mad-2 reduce signal to 14% and 10% of wild type, respectively. Error bars in all graphs represent SEM.