FIGURE 6.

And-1 associates with CENP-A when cells transit from mitosis to G₁ phase. A, schematic showing synchronization of cells in mitosis and release into late mitosis and early G₁ phases. B, FACS analysis of cells harvested as in A. C, localization of And-1 with CENP-A in late mitosis and early G₁ phase. Representative images show And-1 (red) and CENP-A (green) localization in HCT116 cells during late telophase/early G₁ phase. Insets are enlarged images of co-localization indicated by arrowheads. Cells were preextracted with 0.3% Triton X-100. D, quantification of cells with And-1 and CENP-A overlapping or adjacent signals in HCT116 cells for each time point evaluated after release into G₁ (n = 60–150 cells for each time point). E, interaction between And-1 and CENP-A during late telophase/early G₁ phase. Left, chromatin-free extracts from 293T cells co-transfected with FLAG-And-1 and YFP-CENP-A treated as in A, followed by immunoprecipitation with anti-FLAG and immunoblotting with antibodies as indicated. Right, quantification of FLAG-And-1-associated YFP-CENP-A as in left panel. The signals of FLAG-And-1-associated YFP-CENP-A and FLAG-And-1 immunoprecipitations (IP) were quantified by Scion imaging software, and the relative levels of And-1-associated YFP-CENP-A are YFP-CENP-A signal-normalized by FLAG-And-1 immunoprecipitation signals. Data represent the mean ± S.D. (error bars) from three independent experiments. F, enrichment of And-1 at α-satellite repeat (Satα) region. Cells treated as in A were harvested for ChIP assay as in Fig. 3B. y-axis represents the relative enrichment of the And-1 proteins at the α-satellite repeat region at centromere compared with the IgG control (after normalization with input precipitations, data ± S.D., n = 3).