Identification of the CENP-S–associated protein CENP-X. (A) Immunoblots of DT40 cell extracts fractionated on a Superose 6 gel filtration column using antibodies against anti–CENP-O or –CENP-S. Signal intensities are plotted in the graph, and peak fractions are indicated by arrowheads. (B) SDS-PAGE of proteins isolated by IP with anti-Flag antibodies using cells in which expression of CENP-P or -S was replaced with CENP-P–Flag or CENP-S–Flag, respectively. Wild-type (WT) DT40 cells were also used for IP with anti-Flag antibodies as a control. (C) High sensitivity mass spectrometric analysis of the purifications of chicken and human centromere proteins indicating the percentage sequence coverage for each polypeptide. (D) Co-IP of CENP-S with CENP-X. Immunoprecipitates of CENP-X–expressing and wild-type cells with anti-Flag antibodies were separated by SDS-PAGE and analyzed by Western blotting with anti-Flag or –CENP-S antibodies. (E) Localization of GFP-tagged CENP-X throughout the cell cycle in DT40 cells. Centromeres were costained with anti–CENP-C antibodies. M, molecular mass marker. Bar, 10 µm.