FIGURE 2.

And-1 is required for the localization of CENP-A at centromeres. A, And-1 depletion compromises the localization of CENP-A at centromeres. HCT116 cells treated as in Fig. 1A were fixed 72 h after siRNA transfection, and CENP-A was detected with α-CENP-A monoclonal antibody (green). Centromeres were identified with an anti-CENP-B (red) antibody. Scale bar, 10 μm. B, intensity of endogenous CENP-A at centromeres in siGl2- or siAnd-1-treated cells is shown. Data are means ± S.D. (error bars); n, number of cells analyzed. C, And-1 depletion mildly reduces CENP-A chromatin association. HCT116 cells treated as in Fig. 1A were harvested for chromatin binding assays or whole cell extraction (WCE) immunoblotting. D, And-1 depletion by siRNA targeting the 3′-UTR of And-1 causes a reduction of the localization of CENP-A at centromeres in HCT116 cells expressing FLAG alone but not in cells expressing FLAG-And-1 from cDNA. The quantification of CENP-A signals is in the right panel.