2.5. In Vitro Tubulin Polymerization Assay

The fluorescence-based tubulin polymerization assay was conducted using the Tubulin Polymerization Assay Kit from Cytoskeleton (Cat. #BK011P; Cytoskeleton, Denver, CO) following the manufacturer’s protocol. The reaction was performed in a final volume of 10 μL of PEM buffer (80 mM PIPES, 0.5 mM EGTA, 2 mM MgCl₂, pH 6.9) containing 2 mg/mL bovine brain tubulin, 10 μM fluorescent reporter, and 1 mM GTP in a 96 well plate at 37 °C. OAT-449 was added to 3 μM final concentration, and 500 μM CaCl₂ or 3 μM paclitaxel were tested as positive and negative controls for the inhibition or enhancement of tubulin polymerization, respectively. Tubulin polymerization was determined by measuring the fluorescence emission at λ = 420 nm (excitation λ = 360 nm) for 1 h at 1 min intervals using a Flexstation 3 microplate reader (Molecular Device LLC, Sunnyvale, CA, USA). The data were analyzed using Soft Max Pro 4.4.1. software (Molecular Device LLC, Sunnyvale, CA, USA).