Fig. 1. CENP-A is targeted to the kinetochore region of centromeres and, at higher levels of expression, along chromosome arms. (A) HA-tagged CENP-A was immunolocalized with anti-HA antibody (red) in a stable HeLa cell line that expresses the gene in trans from a CMV-based tetracycline-regulated promoter (Shelby et al., 1997). Cells were arrested in mitosis with Colcemid and harvested by mitotic shake-off. Centromeres were detected using SH-CREST auto-antiserum (green), which recognizes antigens in both the pairing and kinetochore domains of centromeres. DNA was counterstained with DAPI (blue). Bar, 1 µm. (B) HA-epitope-tagged human CENP-A cDNA was re-transfected transiently into the stable HeLa cell line. Under these conditions, HA-tagged CENP-A was found to target the lengths of chromosomes (red). Following 
24 hours of expression, CENP-A-(HA) was predominately found on the euchromatic arms of chromosomes, with the pericentric heterochromatin of certain chromosomes containing reduced levels of staining (arrows). Phosphorylated histone H3 was detected using a specific antibody (green) and was found to be interspersed with the mistargeted CENP-A along the arms and within the pericentric heterochromatin of all chromosomes. Bar, 10 µm.
