General laboratory chemicals were obtained from Merck and Thermo Fisher Scientific. Commercially available polyclonal antibodies (pAbs) or monoclonal antibodies (mAbs) were used for pT288 Aurora A (rabbit mAb; 3079S; Cell Signaling Technology), Aurora A (for immunoblotting: rabbit mAb; 4718S; Cell Signaling Technology), Phospho–Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198; rabbit mAb, 2914S; Cell Signaling Technology), Aurora B (mouse mAb, 611083; BD Transduction labs), pT232 Aurora B (rabbit pAb, Rockland, 600-401-677), PPP6C (rabbit, mAb, ab131335; Abcam), NDC80 pS55 (for immunoblotting: rabbit pAb, GeneTex, GTX70017, batch no 42774), NDC80 (for immunoprecipitation and immunoblotting: mouse mAb, Santa Cruz, sc-515550; for immunoprecipitation only: rabbit pAb, ProteinTech, 18932-1-AP and mouse mAb, Abcam, ab3613; for immunofluorescence: mouse mAb [clone 9G3], ab3613, Abcam), MPS1 (rabbit pAb, 10381-1-AP; ProteinTech), Cyclin B1 (mouse mAb; 05-373; Millipore), pT320 PPP1CA (rabbit mAb, ab62334; Abcam), PPP1CA (rabbit pAb, A300-904A; Bethyl Laboratories), pT481 PRC1 (rabbit mAb, ab62366; Abcam), tubulin (mouse mAb; T6199; Merck), BUB1 (rabbit pAb, Bethyl Laboratories, A300-373A), BUBR1 (mouse mAb, Merck, MAB3612), CENP-A (mouse mAb, ab13939; Abcam; mouse mAb, GTX13939; GeneTex), CENP-C (guinea pig pAb, PD030; MBL life science), CREST (human pAb, 15-234; Antibodies Inc.), Mab414 (nuclear pore proteins [NUPs]; mouse mAb, ab24609; Abcam), NUP153 (rabbit pAb, A301-788A; Bethyl Laboratories), TPX2 (mouse mAb, ab32795; Abcam), MAD1 (rabbit pAb, GTX105079; GeneTex), pericentrin (rabbit pAb, ab4448; Abcam), pT210 PLK1 (mouse mAb, BD-558400; BD Biosciences, rabbit mAb, ab155095; Abcam, rabbit pAb, 5472S; Cell Signaling Technology), Nuf2 (mouse mAb, sc-271251; Santa Cruz) and GFP (mouse mAbs, 11814460001; Roche). Antibodies against Aurora A (sheep pAb), Astrin (rabbit, pAb), Kinastrin, (sheep, pAb), mCherry (sheep, pAb), GFP (sheep, pAb), and PLK1 (goat, pAb) have been described previously (Bastos and Barr, 2010; Dunsch et al., 2011; Neef et al., 2005; Thein et al., 2007; Zeng et al., 2010).

NDC80 phosphoantibodies were raised to Aurora sites described in the literature (Kettenbach et al., 2011). Custom rabbit pAb anti-pS62 and anti-pT61-pS62 NDC80 were raised by Eurogentec using a 28-d protocol. For immunizations, peptides CLFGKRT-S(PO3H2)-GHGSRN and CSLFGKR-T(PO3H2)-S(PO3H2)-GHGSRN fused to KLH carrier protein were used, respectively. Total IgG was isolated from postimmune sera by affinity purification on Sepharose-protein G (17-0618-05; Merck, Cytiva). Custom sheep pAb anti-pS55 and anti-pS44 NDC80 were raised by the Scottish Blood Transfusion Service using a 4-mo-long program, and peptides CSERKVS(PO3H2)LFGKR and CFGKLS(PO3H2)INKP, respectively, were fused to KLH carrier protein for immunizations. Total IgG was isolated from postimmune sera by affinity purification on Sepharose-protein G. NDC80 pS55 and pS44 specific antibodies were further immunopurified by incubation with the phosphopeptide antigen immobilized on Sulfo-Link Coupling Gel (20401; Pierce). Secondary donkey antibodies against mouse, rabbit, or sheep, labeled with HRP were purchased from Jackson ImmunoResearch Laboratories (715-035-150, 711-035-152, 713-035-147, respectively). Secondary antibodies for immunofluorescence were purchased from Thermo Fisher Scientific and Jackson ImmunoResearch Laboratories.