Tubulin polymerization assay

Tubulin polymerization was assessed with the use of purified porcine tubulin purchased from Cytoskeleton Inc. (Denver, CO, USA) in accordance with a protocol recommended by the manufacturer. Tubulin was dissolved in a buffer containing: 80 mM PIPES pH 6.9, 2 mM MgCl₂, 0.5 mM EGTA and 1 mM GTP, at a final concentration of 3 mg/mL and placed in a 96-well plate (0.3 mg per well). The polymerization reaction was started by increasing temperature from 4 to 37 °C upon transfer of the reaction mixture to a pre-warmed plate. The assembly of microtubules was monitored spectrophotometrically by measuring absorbance at 350 nm for 60 min, at a temperature of 37 °C. Paclitaxel was used as stabilizing positive control.