Cells were lysed in ice-cold radioimmunoprecipitation assay (RIPA) or NEK extraction buffer (NEB) lysis buffer [50 mM tris-HCl (pH 8), 150 mM NaCl, 1% (v/v) Nonidet P-40, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 5 mM NaF, 5 mM β-glycerophosphate, ribonuclease (30 μg/ml), deoxyribonuclease I (30 μg/ml), 1× protease inhibitor cocktail, and 1 mM phenylmethylsulfonyl fluoride (PMSF)] and subjected to SDS-PAGE and analysis by Western blotting. Primary antibodies were rabbit NEK6 (1 μg/ml) (32), goat NEK7 (1:250; Aviva Systems Biology), rabbit NEK9 (0.4 μg/ml; Atlas Antibodies), mouse α-tubulin (0.3 μg/ml; Sigma), mouse acetylated tubulin (1:2000; Sigma), rabbit glyceraldehyde-3-phosphate dehydrogenase (1:500; Cell Signaling Technology), rabbit EML4 (N terminus, A301-908A; C terminus, A301-909A; both 1:500; Bethyl Laboratories), rabbit green fluorescent protein (GFP; 0.5 μg/ml; Abcam), mouse pHistone H3 (1:1000; Abcam), and mouse cyclin B1 (0.5 μg/ml; Santa Cruz Biotechnology). Secondary antibodies were horseradish peroxidase–labeled secondary antibodies (1:1000; Amersham).

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