Antibody Immunofluorescence and Western Blot Analysis

IF primary: rabbit anti-CENP-A (Active Motif, #39713, 1:1000); guinea pig anti-CENP-C^{31 (link)} (1:1000); rabbit anti-ATPsyn-α (Abcam #ab151229, 1:100); mouse anti-ATPsyn-β (Abcam #ab14730, 1:200); goat anti-ATPsyn-γ (Abcam #ab190310, 1:200); rat anti-mCherry (Chromotek 5F8 1:500); mouse anti-tubulin (Sigma DM1A, 1:100); guinea pig anti-MEI-S332^{32 (link)} (gift from T. Orr-Weaver 1:500) and rabbit anti-GFP (Santa Cruz SC-8334). Guinea pig anti-ATPsyn-βlike antibodies (1:200) were generated by co-injection of two KLH and BSA conjugated peptides CKTDAELVKKKDE (amino acid 68–80) and GDAPPAKAEAKKDEK (amino acid 575–587) marked on Supplementary Fig. 1D. IF secondary: Alexa-488, -546, -647-coupled goat anti-mouse, goat anti-rabbit or goat anti-guinea pig (Life Technologies, 1:500). Western analysis: rabbit anti-CENP-A (Active Motif, #39713, 1:1000), mouse anti-ATPsyn-α (Abcam #ab14748, 1:1000), mouse anti-ATPsyn-β (Abcam #14730, 1:1000), rat anti-GST (Chromotek 6G9, 1:1000), mouse anti-red (Chromotek 6G6, 1:1000), mouse anti-poly-his (Sigma Aldrich #H1029, 1:1000), mouse anti-tubulin (Sigma Aldrich #T5168, 1:10,000) and rabbit anti-histone 3 (Millipore 17–10,254; 1:50,000); guinea pig anti-ATPsyn-βlike (1:1000). Uncropped scans of blots are provided in Supplementary Figure 5.