FIG. 6.
CENP-50 interacts with the CENP-H/CENP-I complex. (A) Localization of CENP-50-GFP (green) in CENP-H, CENP-I, Nuf2, or Hec1 conditional knockout cells. Conditional knockout cells were cultured in the absence (-tet) or presence (+tet) of tetracycline. Expression of each target protein was blocked completely after addition of Tet, as described previously. DNA was counterstained with DAPI (blue). (B) Immunofluorescence analysis of wild-type and CENP-50-deficient cells stained with anti-CENP-A, anti-CENP-C, anti-CENP-H, anti-CENP-I, anti-Hec1, or anti-Nuf2 antibody. Antibody signals were detected with Cy3-conjugated antibodies (red). DNA was counterstained with DAPI (blue). (C) Coimmunoprecipitation of centromere proteins with CENP-50-FLAG. A cell line (F3) in which expression of CENP-50 was replaced with that of CENP-50-FLAG was created. The chromatin fraction of wild-type (Cl18) and F3 (F) cells was immunoprecipitated with anti-FLAG antibody. Immunoprecipitates were separated by SDS-PAGE and analyzed by Western blotting with anti-CENP-50, anti-CENP-H, anti-CENP-I, and anti-Hec1 antibodies.