Microtubules were assembled from 40 μM tubulin in the presence of 5 mM GTP in MRB80 for 1 hour at 37°C and diluted 1:3 in MRB80 and 2 μM Taxol. Three-fifth of the sample was treated with subtilisin (25 μg/ml; P5380, Sigma), and the remainder was left untreated and incubated at 37°C for 10 min. To terminate digestion, 5 mM PMSF and complete protease inhibitors (Roche) were added, and the reaction mix was loaded onto a 30% sucrose cushion and centrifuged in a TLA55 rotor at 100,000g for 45 min at 30°C. The microtubule pellets were resuspended in BRB25 [25 mM Pipes (pH 6.8), 1 mM MgCl2, 1 mM EGTA, and 2 mM DTT] supplemented with complete protease inhibitors and 2 μM Taxol and incubated with EML4(1-207)-Avi on ice for 15 min before pelleting through a 30% sucrose cushion in a TLA55 rotor at 100,000g for 45 min at 4°C. The pellet was taken up in equal volume to supernatant, and samples were analyzed on 12% SDS-PAGE gels, stained with Instant Blue (Expedeon), imaged on a G:BOX (Syngene), and quantified using ImageJ.

For microtubule sedimentation assays, cells were lysed at room temperature in RIPA lysis buffer, including 5 mM NaF and 5 mM β-glycerophosphate. Samples were loaded on the top layer of 30% sucrose in tubulin stabilization buffer [TSB; 1 mM EGTA, 5 mM MgCl2, and 80 mM Pipes (pH 7.0)] before centrifugation at 100,000g for 40 min at 21°C. Supernatants were collected, and pellets were washed with TSB for 10 min at 21°C. Pellets were diluted into a volume equal to that of the supernatants. Samples were subjected to SDS-PAGE and analysis by Western blot.

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