Fig. 2. CENP-B is not recruited to non-centromeric regions of chromatin containing mistargeted CENP-A. HA-tagged human CENP-A cDNA was transiently transfected and overexpressed in HeLa cells. Cells were arrested in mitosis with Colcemid for 3 hours following 48 hours of CENP-A-(HA) expression and were harvested by mitotic shake-off. SH-CREST antiserum, which recognizes CENP-A, was localized throughout the mitotic chromosomes of transfected cells. CENP-B was immunolocalized using a specific antiserum. Anti-CENP-B antibody stained exclusively the centromeres of chromosomes in HeLa cells overexpressing CENP-A. DNA was counterstained with DAPI. Bar, 10 µm.