Analysis of BUB-1 interaction-defective MAD-1 mutants in vivo. (A) GFP::MAD-1 variants expressed from single-copy transgene insertions (Fig. S2 F), propagated in a mad-1Δ background, and analyzed by anti–MAD-1 immunoblotting. α-Tubulin (α-tub) serves as a loading control. (B and C) Images and quantification of GFP::MAD-1 variant localization to unattached kinetochores. Unpaired t tests show that MAD-1^3A is significantly reduced relative to MAD-1^WT (P < 0.0001) and MAD-1^P504 (P = 0.0004); MAD-1^D423A is also significantly reduced (P < 0.0001 relative to MAD-1^WT, and P = 0.0066 relative to MAD-1^P504). The WT value is reproduced from Fig. 2 A. n is number of embryos analyzed. Error bars are the 95% confidence interval of the mean. (D) Immunoblotting of untagged MAD-1 variants. α-Tubulin serves as a loading control. (E) Quantification of time from NEBD to onset of cortical contractility in the presence of monopolar spindles. n is number of embryos analyzed. The red dotted line indicates mitotic duration in mad-1(RNAi), where the checkpoint is inactive. Error bars are the 95% confidence interval of the mean. (F) MAD-1 accumulation between separating homologous chromosomes in early anaphase of oocyte meiosis I. Time (seconds) on the bottom left of merge images are relative to anaphase onset. Images on the bottom left show that this localization depends on BUB-1. Images on the bottom right show loss of this localization for MAD-1^3A but not MAD-1^P504A. 3–11 embryos were imaged per condition. Ctrl, control. (G) Nuclear periphery enrichment of MAD-1 for the indicated conditions. Dotted line indicates the embryo outline. More than eight embryos were imaged per condition. Bars, 5 µm.