Figure S4.
Metaphase HJURP centromere localization and CENP-A assembly require CENP-C. (a) Representative Western blot of CENP-C depleted extract samples used in (b) and (c). Depletion (Δ) is indicated above each column. A Tubulin Western blot is included as a loading control. (b) Dual depletion of CENP-C and M18BP1 in metaphase prevents localization of HJURP mutants. Quantification of FLAG-HJURP centromere localization on sperm chromatin in M18BP1 and/or CENP-C depleted metaphase and interphase extracts. Values are normalized to wild-type FLAG-HJURP centromere signal in mock-depleted interphase extract. Bottom rows indicate depletion (Δ) status (M18BP1, CENP-C, or IgG antibody) for each condition. Plot shows mean FLAG-HJURP signal on sperm chromatin ± SEM of at least three experiments (**, P < 0.01; Wilcox/Mann–Whitney Test). (c) CENP-C is required for premature CENP-A assembly in metaphase driven by HJURPS220A or P221A and M18BP1 depletion. Quantification of V5-CENP-A assembly on sperm chromatin in single depletion (M18BP1) interphase and metaphase extracts, and dual depleted (CENP-C and M18BP1) extracts. Values are normalized to V5-CENP-A assembly signal in wild-type HJURP condition on mock-depleted interphase extracts. Bottom rows indicate depletion (Δ) status (M18BP1, CENP-C, or IgG antibody) for each condition. Plot shows mean V5-CENP-A assembly signal on sperm chromatin ± SEM of at least three experiments (*, P < 0.05; ***, P < 0.001; Wilcox/Mann–Whitney Test). (d) Quantification of anti–CENP-C Western blots following CENP-C immunoprecipitation (IP; Fig. 5 c) shows consistent levels of CENP-C in all conditions. Signal was normalized to mock-depleted extract with HJURP S220A and no M18BP1.S addback. (e) Quantification of anti-HJURP Western blots following CENP-C immunoprecipitation (Fig. 5 c) shows M18BP1.S161-580 competes for CENP-C binding with HJURPS220A or HJURPP221A. Signal was normalized to mock-depleted extract with HJURPS220A and no M18BP1.S addback. (f) Quantification of anti–CENP-C Western blots following CENP-C immunoprecipitation (Fig. 5 d) shows consistent levels of CENP-C in all conditions. Signal was normalized to M18BP1-depleted extract with HJURP S220A and no M18BP1.S161-580 addback. (g) Quantification of anti-HJURP Western blots following CENP-C immunoprecipitation (Fig. 5 d) shows that M18BP1.S161-580 competes for CENP-C binding, but the SANTA domain mutant of M18BP1.S that cannot bind to CENP-C fails to compete. Signal was normalized to M18BP1-depleted extract with HJURP S220A and no M18BP1.S161-580 addback. Source data are available for this figure: SourceData FS4.

Metaphase HJURP centromere localization and CENP-A assembly require CENP-C. (a) Representative Western blot of CENP-C depleted extract samples used in (b) and (c). Depletion (Δ) is indicated above each column. A Tubulin Western blot is included as a loading control. (b) Dual depletion of CENP-C and M18BP1 in metaphase prevents localization of HJURP mutants. Quantification of FLAG-HJURP centromere localization on sperm chromatin in M18BP1 and/or CENP-C depleted metaphase and interphase extracts. Values are normalized to wild-type FLAG-HJURP centromere signal in mock-depleted interphase extract. Bottom rows indicate depletion (Δ) status (M18BP1, CENP-C, or IgG antibody) for each condition. Plot shows mean FLAG-HJURP signal on sperm chromatin ± SEM of at least three experiments (**, P < 0.01; Wilcox/Mann–Whitney Test). (c) CENP-C is required for premature CENP-A assembly in metaphase driven by HJURPS220A or P221A and M18BP1 depletion. Quantification of V5-CENP-A assembly on sperm chromatin in single depletion (M18BP1) interphase and metaphase extracts, and dual depleted (CENP-C and M18BP1) extracts. Values are normalized to V5-CENP-A assembly signal in wild-type HJURP condition on mock-depleted interphase extracts. Bottom rows indicate depletion (Δ) status (M18BP1, CENP-C, or IgG antibody) for each condition. Plot shows mean V5-CENP-A assembly signal on sperm chromatin ± SEM of at least three experiments (*, P < 0.05; ***, P < 0.001; Wilcox/Mann–Whitney Test). (d) Quantification of anti–CENP-C Western blots following CENP-C immunoprecipitation (IP; Fig. 5 c) shows consistent levels of CENP-C in all conditions. Signal was normalized to mock-depleted extract with HJURP S220A and no M18BP1.S addback. (e) Quantification of anti-HJURP Western blots following CENP-C immunoprecipitation (Fig. 5 c) shows M18BP1.S161-580 competes for CENP-C binding with HJURPS220A or HJURPP221A. Signal was normalized to mock-depleted extract with HJURPS220A and no M18BP1.S addback. (f) Quantification of anti–CENP-C Western blots following CENP-C immunoprecipitation (Fig. 5 d) shows consistent levels of CENP-C in all conditions. Signal was normalized to M18BP1-depleted extract with HJURP S220A and no M18BP1.S161-580 addback. (g) Quantification of anti-HJURP Western blots following CENP-C immunoprecipitation (Fig. 5 d) shows that M18BP1.S161-580 competes for CENP-C binding, but the SANTA domain mutant of M18BP1.S that cannot bind to CENP-C fails to compete. Signal was normalized to M18BP1-depleted extract with HJURP S220A and no M18BP1.S161-580 addback. Source data are available for this figure: SourceData FS4.

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