In vitro tubulin polymerization assay

To determine tubulin polymerization in vitro, a Tubulin Polymerization Assay kit (Cytoskeleton, Denver, CO, USA) was used as per the manufacturer’s instructions. Briefly, a reaction mixture containing (1) tubulin, (2) GTP, (3) Buffer 1, and (4) Tubulin Glycerol Buffer were mixed and kept on ice. Then, the respective aliquot of 5 μl of ITC in deionized H₂O (final concentration 40 μM) was added in a 96-well plate, and warmed for 1 min at 37°C in an incubator. Tubulin polymerization was initiated by adding 50 μl of reaction mixture and monitored for fluorescent intensity (E_x=360 nm; E_m=420 nm). Measurement was recorded using a Varioska Flash spectral scanning multimode reader, Thermo Fisher Scientific (San Jose, CA, USA). Readings were taken in the kinetic mode for 1 h (for a total of 61 readings).