Nocodazole

Centrosome purification was performed as previously described with some modifications^{51 (link)}. U2OS cells that had been treated with a solution containing 10 μg/ml nocodazole (Santa Cruz Biotechnology, CA, USA) and 5 μg/ml cytochalasin B (Sigma-Aldrich, St. Louis, MO, USA) for 1.5 h were lysed with lysis buffer (1 mM HEPES [pH 7.2], 0.5% NP-40, 0.5 mM MgC1₂, 0.1% β-mercaptoethanol and protease inhibitors) on ice for 20 min. Swollen nuclei and chromatin aggregates were removed by centrifugation (2500 × g, 10 min). The supernatant was supplemented with HEPES buffer to a final concentration of 10 mM and incubated with 2 Units/ml of DNase I (Sigma-Aldrich, St. Louis, MO, USA) on ice for 30 min. The lysate was underlaid with 1 ml of 60% sucrose solution and centrifuged at 25,000 × g for 30 min at 4 °C. The crude centrosome preparation was diluted with lysis buffer and layered onto a discontinuous sucrose gradient (from bottom to top, containing 0.5, 0.3, and 0.3 ml of 70%, 50%, and 40% sucrose solutions, respectively) in a 5 ml tube, followed by centrifugation at 97,000 × g for 1.5 h at 4 °C. Subsequently, fractions were collected, diluted with 10 mM PIPES (pH 7.2), and centrifuged at 14,000 rpm for 10 min to pellet the centrosomes. The centrosome pellets were resuspended in 40 µl of SDS sample buffer.