The SAH domain is required for CPC localization and the SAC in Xenopus. (A) Top, diagram of INCENP constructs. CEN, centromere-targeting domain; IN, IN-box; HP1, HP1-binding motif. Bottom, Xenopus egg extracts were either mock depleted (Mock) or depleted of CPC (ΔCPC); reconstituted with xAurora B, xDasra A, xSurvivin, and FL or ΔSAH GFP-INCENP; and maintained at M-phase with CSF. After addition of high concentrations of sperm and nocodazole, CSF was inactivated by calcium to monitor SAC-mediated maintenance of M phase. Time after calcium addition is indicated. Presence of H3T3ph, monitored by Western blot, marks M phase. (B) Immunofluorescence of GFP-xINCENP on sperm chromatin in M-phase extract treated with nocodazole. Green, GFP; purple, DAPI. Bar, 5 µm. (C) Immunofluorescence of GFP-xINCENP (green) on preformed CENP-A chromatin arrays (purple) in M-phase extracts. Bar, 6 µm. (D) Immunofluorescence of GFP or GFP-xSAH on sperm chromatin in M-phase extracts treated with nocodazole. Median, interquartile range (IQR; box), and ±1.5 × IQR (error bars) are shown. Two-tailed Mann–Whitney t test; n = 20 spreads per sample; ***, P ≤ 0.001. Bar, 2 µm.