Constitutive impairment of Mad1–Megator interaction reduces the levels of C-Mad2 at KTs. (A and B) Western blots (A) and quantifications (B) of Megator, Mad1, and Mad2 protein levels in lysates from control and Megator-depleted S2 cells expressing Megator-EGFP transgenes. The graph represents the signal intensities of Megator, Mad1, and Mad2 relative to tubulin from at least two independent experiments. The mean value for control parental cells was set to 1. (C and D) Immunofluorescence images (C) and quantifications (D) of C-Mad2 at unattached KTs of S2 cells depleted of endogenous Megator and expressing Megator-EGFP transgenes (n ≥ 148 KTs). (E and F) Immunofluorescence images (E) and quantifications (F) of C-Mad2 levels at unattached KTs of control and Megator-depleted S2 cells (n ≥ 224 KTs). (G) Mitotic timings of control and Megator-depleted S2 cells expressing Megator-EGFP transgenes under unperturbed (absence of spindle poisons) conditions (n ≥ 11 cells). (H) Immunoprecipitates (IP) of BubR1 from lysates of control and Megator-depleted S2 cells expressing Megator-EGFP transgenes. When indicated, cells were incubated with colchicine for 18 h. The graph represents the ratio between the signal intensities of Cdc20 and BubR1 present in BubR1 IPs from two independent experiments. The values for control parental cells were set to 1. In B, data are presented as mean ± SEM; in D, F, G, and H, data are presented as mean ± SD. *, P < 0.05; ****, P < 0.0001 (Kruskal–Wallis, Dunn’s multiple comparison test in D and Student’s t test in F and G). Scale bars, 5 µm (inset, 0.5 µm).