Microtubule Enrichment and Analysis

This was performed as described previously^{28 (link)} with minor modifications. Briefly, cells synchronized and enriched in mitotis were lysed at 4°C in 100 mM 1,4-piperazinediethanesulfonic acid (PIPES), at pH 6.8, 1 mM MgCl₂, 2 mM ethylene glycol tetra acetic acid (EGTA) and 1% Triton X-100, and spun at 13 000 × g for 30 min. To the supernatant thus obtained, purified tubulin (Cytoskeleton, Denver, CO, USA; 4 μg), dithiothreitol (1 mM), guanosine-5′-triphosphate (GTP; 1 mM) and taxol (Sigma-Aldrich) (10 μM) were added to cleared lysates, and incubated for 1 h at 37°C. Nocodazole (100 μM) was added as a negative control. Lysates were layered over a 20% sucrose cushion in the above buffer and spun at 48 000 r.p.m. for 1 h at room temperature (rotor TLA 120.1, Beckman Coulter). Microtubule pellets were collected after removing lysate and cushion, bound proteins were separated by SDS-PAGE and analyzed by western blotting.