2.6. Immunofluorescence Staining

To detect acetyl-α-tubulin expression after treatment with the compounds, MDA-MB-231 cells treated with GM-90257 and GM-90631 were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.1% triton-X 100 in PBS. After blocking with 0.1% bovine serum albumin, the cells were stained with primary antibodies against acetyl-α-tubulin (Sigma). To analyze the co-localization of αTAT1 and α-tubulin, MDA-MB-231 cells transfected with Dsred2-αTAT1 were prepared in the same manner and stained with primary antibodies against α-tubulin (Sigma). To stain the microtubule bundles shown, soluble tubulin proteins were extracted by dipping into 0.1% Triton X-100 for 15 s before fixation. Cells were fixed with ice-cold methanol at −20 °C for 5 min, after which the samples were treated with blocking solution (0.1% bovine serum albumin in PBST) for 1 h. The cells were stained with primary antibodies against α-tubulin (Sigma). All fluorescence microscopy images were obtained using a confocal microscope (FV1000; Olympus, Tokyo, Japan).