The SAH domain is required for CPC localization at the centromere in human cells. (A) Western blot of HeLa cell extracts reconstituted as indicated. Anti–Aurora A/B/Cph detects the phosphorylated T-loop of Aurora A and Aurora B. (B and C) Integrated intensity at the centromere was quantified for each epitope in taxol (B) or nocodazole (C). Data represent n ≥ 28 cells in B and n ≥ 74 cells aggregated from three independent experiments for C. Median, IQR (box), and ±1.5 × IQR (error bars) are shown. Two-tailed Mann–Whitney t test; **, P ≤ 0.01; ***, P ≤ 0.001. Bar, 2 µm. AU, arbitrary units. (D and E) FRAP of centromeric foci in taxol-treated (D) or nocodazole-treated (E) cells reconstituted with hINCENP or hINCENP ΔSAH. The mean FRAP recovery curve with a 95% confidence interval is displayed with the mean t1/2 ± SD for each sample; data compiled from n = 2 independent experiments per condition, n ≥ 30 centromeres for each condition.
