Tubulin polymerization assay was performed essentially as previously described [29, 54] . Briefly, cells grown to confluency in 24-well plates were washed twice with 1X PBS. To separate polymerized (P) from soluble (S) tubulin, the cells were all incubated at 37uC for 5 min in the dark in hypotonic lysis buffer containing 5 mM paclitaxel, 10 mM Trichostatin-A (Calbiochem, San Diego, CA), 1 mM MgCl2, 2 mM EGTA, 0.5% Nonidet P-40, 2 mM phenylmethylsulfonyl fluoride, 200 units/ml aprotinin, 100 mg/ml soybean trypsin inhibitor, 5.0 mM e-amino caproic acid, 1 mM benzamidine, and 20 mM Tris-HCl, pH 6.8, vortexed vigorously and centrifuged at ,15,000 g at 22uC for 10 minutes. The supernatants containing soluble 'S' tubulin were transferred to another Eppendorf tube separating them from the pellets containing polymerized 'P' tubulin. Upon separation, tubes were placed on ice and pellets of polymerized 'P' tubulin were resuspended by sonication for 10-20 seconds in a volume of lysis buffer equal to the soluble 'S' fraction. Each had gel sample buffer added, equal aliquots were separated by 10% SDS-PAGE, and western blots using anti-a-tubulin antibody were obtained. The immunoblots were scanned, and densitometric analysis was performed using the public domain NIH Image program ImageJ (available on the Internet at http://rsb.info.nih.gov/nih-image/). The percentage of polymerized 'P' tubulin was determined by dividing the densitometry value of polymerized 'P' tubulin by the total tubulin content (the sum of the densitometry values of soluble 'S' and polymerized 'P' tubulin). An advantage of this assay is that the amount of total protein loaded for each sample is irrelevant since the 'P' and 'S' fractions are equalized for each pair, and it is the proportion of the polymerized to the soluble tubulin fraction that is measured.