Figure 3.

MDs bind to colchicine site of β-tubulin. A, porcine brain tubulin (1 μm) were treated with 1 μm SKLB028, 1 μm vinblastine, 10 μm vinblastine, 1 μm colchicine, 10 μm colchicine, or 1 μm SKLB050 for 1 h before treated with 1 μm Bio-SKLB028 for another 2 h. After totally wash of unbounded compounds and cross-linked by a UV hybridization incubator for 10 min, the samples were blotted for biotin. Total proteins were blotted for β-tubulin as a loading control. B, HCT-8/V cells were incubated with or without indicated concentrations of MDs for 2 h, and then 100 μm EBI was added to cells and incubated for another 2 h. Total protein was lysed with RIPA lysis buffer and subjected to Western blotting analysis for β-tubulin and GAPDH. Vin, vinblastine; Col, colchicine; Con, control; Tub, tubulin.