Anti β tubulin mouse monoclonal antibody

Hela cells (5 × 104/ well) were plated on coverslips in 6-well plates and treated with compound 5 and Monastrol at 20 µM for 24 h. Monastrol was used as a positive control, while 0.1% DMSO was used as a negative control. The cells were rinsed with PBS, fixed with 3.7% paraformaldehyde, and permeabilized with 0.1% Triton X-100. The cells were blocked with 1% BSA in PBS for 1 h prior to incubation with anti-β-tubulin mouse monoclonal antibody (#86,298, Cell Signaling, San Francisco, CA, USA) overnight at 4 °C. The cells were washed with PBS for 1 h in the dark, and then incubated with Alexa Fluor® 488 secondary antibodies (Abcam). The cellular microtubules were observed with a fluorescence microscope (Olympus BX43, Japan).

The MCF-7 cancer cells plated on coverslips on six-well plate (1 × 10⁵/well) were treated with the indicated concentration of control, colchicine (3 μM), paclitaxel (3 μM), and the test compound 16b (5 μM) for 24 h as previously reported²³ (link). After treatment, cells were rinsed twice with PBS, fixed with 3.7% paraformaldehyde, and permeabilised with 0.1% Triton X-100. Cells then were blocked with 1% BSA in PBS for 1 h before further incubation with anti-β-tubulin mouse monoclonal antibody overnight at 4 °C (#86298, Cell Signaling, San Francisco, CA). Cells were incubated with Alexa Fluor^® 488 secondary antibodies (Abcam, Cambridge, UK), after being washed with PBS for 1 h in a darkroom. Cellular microtubules were observed with the Nikon Eclipse Ti microscope (Minato-ku, Japan).