Nxa931 1ml

Mouse anti-GFP (11 814 460 001; Roche), rabbit anti-GTSE1 (custom generated; described in Scolz et al [2012] (link)), and rabbit anti-Aurora kinase B (ab2254; Abcam) were used for Western blots. Mouse anti–α-tubulin (DM1α; Sigma-Aldrich), rabbit anti-Cep135 (custom generated, described in Bird and Hyman [2008] (link)), goat anti-GFP (MPI Dresden; described in Poser et al [2008] (link)), and goat anti-Aurora A (sc-27883; Santa Cruz) were used for both Western blot and immunofluorescence. Donkey anti-goat Alexa 488 (705 545 147; Jackson Immunoresearch), donkey anti-mouse Alexa 594 (A90-337D4; Bethyl Laboratories), and donkey anti-rabbit Alexa 650 (A120-208D5; Bethyl Laboratories) were used in immunofluorescence. Donkey anti-goat HRP (SC-2020; Santa Cruz), sheep anti-mouse HRP (NXA931-1ml; Amersham), and donkey anti-rabbit HRP (NXA934-1ml; Amersham) were used for Western Blots.

HeLa cells were grown in DMEM supplemented with 10% tetracycline-free fetal bovine serum, 1% L-glutamine and 1% Penicillin-Streptomycin at 37°C in a 5% CO₂ atmosphere. Cells were treated with 50 ng/mL doxycycline (Sigma) for 18 hr and then treated with both 50 ng/mL doxycycline and 9 µM CDK1 inhibitor RO-3306 (Merck) for additional 6 hr before harvest. Cells were washed twice with PBS, resuspended in lysis buffer containing 75 mM HEPES pH 7.5, 1.5 mM EGTA, 1.5 mM MgCl₂, 150 mM NaCl, 10% glycerol, 0.1% NP-40, 1 mM PMSF and Protease Inhibitor Mix HP Plus (SERVA), lysed using Bioruptor Plus sonication device (Diagenode) and centrifuged at 16,000 x g for 30 min at 4°C. The supernatant containing 2 mg protein was incubated with 7.5 µl GFP-Trap_A beads (Chromotek) for 2 hr at 4°C. The beads were washed thrice with the lysis buffer containing 300 mM NaCl instead of 150 mM NaCl. Proteins were eluted by adding SDS sample buffer and were analyzed using Tricine-SDS-PAGE and Western blot analysis. EGFP-M18BP1, mCherry-M18BP1, mCherry-Mis18α, Mis18β and vinculin were detected using following antibodies: anti-GFP (Abcam, AB6556), anti-mCherry (Novus, NBP1-96752), anti-Mis18β (Atlas, HPA052271), anti-vinculin (Sigma, V9131) anti-mouse-HRP (Amersham, NXA931-1ML) and anti-rabbit-HRP (Amersham, NXA934-1ML).