FIGURE 3.

And-1 interacts with CENP-A. A, And-1 interacts with YFP-CENP-A. 293T cells co-expressing FLAG-And-1 and YFP or FLAG-And-1 and YFP-CENP-A were harvested to prepare chromatin-free fractions for co-immunoprecipitation assays. FLAG-And-1 immunoprecipitates were resolved by SDS-PAGE and immunoblotted for the indicated proteins. B, And-1 specifically associates with centromere. And-1 protein levels at replication origin BG40.9 or surrounding region BG72, α-satellite repeat (Satα) or pericentromeric region (Sat2) are shown. IgG was used as the measurement of nonspecific chromatin precipitation. Quantitative PCR was used to ascertain the And-1 levels at the indicated chromatin regions. y-axis represents the relative enrichment of the And-1 proteins compared with the IgG control (after normalization with input precipitations, data ± S.D. (error bars), n = 3). C, schematic shows And-1 protein domains and deletion mutants used for protein interactions. D, full-length And-1 and And-1(330–1129) interact with chromatin-free CENP-A. Chromatin-free extracts from 293T cells co-transfected with GFP-CENP-A and FLAG-And-1 or FLAG-And-1 mutants were immunoprecipitated with anti-FLAG and immunoblotted for the indicated antibodies.