Fig. 10. Kinetochore components HZW10 and CENP-E are not mistargeted to ectopic regions with CENP-A. To determine if kinetochore microtubule-associated proteins are recruited to non-centromeric regions of chromatin to which CENP-A has been mistargeted, HA-tagged CENP-A was overexpressed in HeLa cells, and human ZW10 and CENP-E were immunolocalized using a specific antisera. Cells were arrested in mitosis with Colcemid for 3 hours following 48 hours of CENP-A-(HA) expression and harvested by mitotic shake-off. (A) Anti-HZW10 antibody was localized exclusively to kinetochores in mitotic cells overexpressing CENP-A (top cell), as was found in untransfected controls within the same preparations (bottom cell). CENP-A-(HA) was detected using anti-HA antibody. DNA was counterstained with DAPI. (B) CENP-E was similarly localized exclusively to kinetochores in cells overexpressing CENP-A. Ectopic CENP-A and centromeres were detected using SH-CREST antiserum. Bars, 10 µm.