M18BP1.S prevents HJURP association with CENP-C and new CENP-A assembly during metaphase. (a) M18BP1 depletion causes premature HJURPS220A centromere localization in metaphase and new CENP-A assembly. Representative images of sperm nuclei incubated with M18BP1-depleted metaphase or interphase Xenopus egg extracts complemented with the HJURP mutants indicated to the left of the images. Right-hand labels indicate mock-depleted or M18BP1-depleted and M18BP1.S addback for each condition. Metaphase and interphase extracts were supplemented with RNA encoding V5-CENP-A to assay for new CENP-A assembly. Immuno-localized protein is specified above. Insets are magnified 300%. Scale bar represents 5 μm. (b) Quantification of FLAG-HJURP centromere localization in a. Values are normalized to the wild-type FLAG-HJURP centromere signal in mock-depleted interphase extract. Bottom rows indicate antibody depletion (Δ; M18BP1 or IgG antibody) and M18BP1.S addback (+) for each condition. Plot shows mean FLAG-HJURP signal on sperm chromatin ± SEM of at least three experiments (*, P < 0.05; **, P <0.01; n.s. = 0.094; Wilcox/Mann–Whitney Test). (c) Quantification of V5-CENP-A assembly in a. Values are normalized to the V5-CENP-A assembly signal in wild-type HJURP condition in mock-depleted interphase extracts. Bottom rows indicate depletion (Δ) status (M18BP1 or IgG antibody) and M18BP1.S addback (+) for each condition. Plot shows mean V5-CENP-A assembly signal on sperm chromatin ± SEM of at least three experiments (*, P < 0.05; **, P < 0.01; n.s. = 0.14; Wilcox/Mann–Whitney Test).