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. 2015 Sep 14;210(6):899–916. doi: 10.1083/jcb.201506020

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© 2015 Wynne and Funabiki

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Figure 6. — Aurora B is required for the core outer kinetochore, whereas Bub1 is specifically required for the expandable module. (A) Mitotic chromosomes assembled in Xenopus egg extracts immunodepleted of Bub1 kinase (ΔBub1) or the CPC using antibodies recognizing INCENP (ΔINC) and treated with nocodazole. (B) Quantification of the total signal, P values from Mann–Whitney test, and percentage of control mean value are shown. A.U., arbitrary unit. (C) Western blots of total extract (left) and chromosomes purified from nocodazole-treated control (ΔIgG) or CPC-depleted (ΔINC) extracts (right) show a reduction in CENP-C and a more severe loss of Ndc80, Mis12, and Zwint signal. (D) Mitotic chromosomes assembled in nocodazole-treated extracts. (left) Maximum intensity projections of whole nuclei. (right) Single optical section from the same dataset to highlight the inner centromeric staining underlying the expanded kinetochores (yellow arrows). (E) Mitotic chromosomes assembled in indicated extracts. Higher magnification images of a single optical section within the chromosome mass of Dasra-stained samples (area of inset shown with white boxes) are shown in the right-most panels to highlight the mislocalization of the CPC in all Bub1-depleted nuclei (cyan arrows) relative to controls (yellow arrows). wt, wild type. (F) Quantification normalized to the intensity of the BubR1 staining in control depletion (ΔIgG). Mean and standard deviation plotted in black. Mann–Whitney test: ***, P < 0.0001; ns, P = 0.28. All images are maximum intensity projections, except when “single section” is indicated, of 3D-SIM datasets. Bars: (A, D, and E, left) 1 µm; (E, right) 0.2 µm.