Immunofluorescence and Western Blotting Protocols

Immunofluorescence and Western blotting were performed essentially as previously described [84 (link)]. For immunofluorescence, the following antibodies were used. Primary antibodies: anti-Lamin A (1:1000; Sigma-Aldrich), anti-centromere protein (ACA, CREST, 1:400; Antibodies Incorporated), anti-phospho-Histone H3 (Ser10, 1:300; Sigma-Aldrich), anti-α-tubulin DM1A (1:500; Sigma-Aldrich), anti-CENP-A (3–19, 1:300; Invitrogen). Secondary antibodies: Alexa Fluor 647 Goat Anti-Rabbit IgG (H+L, 1:200; Invitrogen), Alexa Fluor 647 IgG (H+L) Cross-Adsorbed Goat anti-Human (1:200; Invitrogen), Alexa Fluor 488 goat anti-rabbit (1:200; Invitrogen), Texas Red-X goat anti-mouse IgG (1:200; Invitrogen) and Alexa Fluor 488 Phalloidin (to label F-actin, 1:1000; Invitrogen). For Western blotting, the following primary antibodies were used: anti-α-tubulin DM1A (1:5000; Sigma-Aldrich), anti-KIF18A (1:1000; Bethyl Laboratories Inc.), anti-CENP-A (3–19, 1:2000; Invitrogen) and anti-Histone H3 (3H1, 1:1000; New England BioLabs).