2.11. Microtubule Polymerization Assay

Cell-free tubulin polymerization assays were conducted according to the manufacturer’s instructions (BK006P; Cytoskeleton, Inc., Denver, CO, USA). Briefly, tubulin proteins (>99% purity) were suspended in G-PEM buffer (80 mM PIPES, pH 6.9, 2 mM MgCl₂, 0.5 mM EGTA, 1 mM GTP) with or without paclitaxel (10 µM; Cytoskeleton, Inc., Denver, CO, USA), colchicine (10 µM; Sigma-Aldrich, St. Louis, MO, USA), and ML111 (0.1 and 1.0 µM) in a 96-well plate, and the absorbance was measured continuously at 340 nm for 60 min (Synergy multimode microplate reader, BioTek, Winooski, VT, USA).