Nek9, Nek6, and Nek7 antibodies were generated in rabbits by Covance against three Nek9 antigens, one Nek6 antigen, and one Nek7 antigen. DNA corresponding with Nek9 amino acids 1–58, 301–349, and 360–388, Nek6 amino acids 1–39, and Nek7 amino acids 1–35 was cloned into pGEX-6p1, expressed in BL21DE3 pLys Escherichia coli, and purified by affinity chromatography using glutathione-Sepharose resin (GE Healthcare). GST was cleaved, antigen solutions were lyophilized, and the three Nek9 antigens were combined and submitted to Covance; the resulting sera was affinity-purified using antigens coupled to N-hydroxysuccinimide ester–Sepharose per Covance protocols.
Lamin A/C antibody was a gift from F. McKeon (University of Houston, Houston, TX). All other antibodies were commercially available as follows: PRC1 and INCENP (Cell Signaling Technology), Mklp1 and GFP (Genetex), Mklp2 (Abcam), Kif14 (Bethyl Laboratories, Inc.), α-tubulin (Sigma-Aldrich), myc (clone 9E10), and citron (BD).
Lamin A/C antibody was a gift from F. McKeon (University of Houston, Houston, TX). All other antibodies were commercially available as follows: PRC1 and INCENP (Cell Signaling Technology), Mklp1 and GFP (Genetex), Mklp2 (Abcam), Kif14 (Bethyl Laboratories, Inc.), α-tubulin (Sigma-Aldrich), myc (clone 9E10), and citron (BD).