Fig. 9. HZwint-1 is mislocalized with CENP-A, but not with CENP-C, during mitosis and interphase. (A) HZwint-1 was immunolocalized in HeLa cultures transiently transfected with HA-tagged CENP-A. Cells were arrested in mitosis with Colcemid for 
3 hours following 48 hours of CENP-A-(HA) expression and harvested by mitotic shake-off. Mitotic cells overexpressing CENP-A, detected with SH-CREST antiserum, demonstrated mislocalization of anti-HZwint-1 along chromosome arms (cell on right). Untransfected cells within the same preparation (cell on left) demonstrated the characteristic staining of anti-HZwint-1 exclusively at kinetochores. DNA was counterstained with DAPI. (B) Cells were grown and stained on coverslips to examine all phases of the cell cycle. Anti-HZwint-1 staining was observed throughout interphase chromatin in cells overexpressing CENP-A (cell on left). Anti-HZwint-1 staining was exclusively cytoplasmic in untransfected cells (cell on right). (C) Overexpression of CENP-C, detected using SH-CREST antiserum, did not result in the mistargeting of HZwint-1 during interphase. Untransfected mitotic cells on the same coverslips were used as a positive control for HZwint-1 staining (data not shown). Bars, 10 µm.
