Monoclonal mouse anti human α tubulin antibody

Generally, cells were harvested 48 h after treatment by trypsin digestion following a washing step with PBS. Cell pellets were lysed using cold radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors followed by 2 s of sonication and a 5-min incubation on ice. After removal of cell debris by centrifugation at 4°C, protein concentrations were determined using the Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Aliquots of cell extracts containing 25–50 μg of protein were resolved by 4%–20% precast polyacrylamide gels purchased from Bio-Rad (Hercules, CA, USA) and imaged using chemiluminescence. The dilution of the various antibodies followed the manufacturers’ instructions. The monoclonal mouse anti-human α-tubulin antibody (Sigma-Aldrich) was added at 4,000× dilution in Tris-buffered saline with Tween 20 (TBST) containing 5% fat-free dry milk. Protein signals on the blot were quantified with the ImageJ program and protein expression was normalized to the SSO control.

Cells were generally harvested 48 h after treatment with trypsin digestion and washed once with PBS. Cell pellets were lysed in cold radioimmunoprecipitation assay buffer (RIPA) containing protease inhibitors, sonicated for 2 s, and placed on ice for 5 min. After removal of the cell debris by centrifugation, protein concentrations were determined using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific). Aliquots of cell extracts containing 25–50 μg of protein were resolved by 10% SDS/PAGE and imaged by an ECL or Odyssey system. The dilution of the various antibodies followed the manufacturers’ instructions. The monoclonal mouse anti-human α-tubulin antibody (Sigma–Aldrich) was added at a 1:4,000 dilution in TBS-Tween containing 5% fat-free dry milk. The secondary ECL anti-mouse IgG-HRP (GE Health Care) whole antibody was added at a 1:7,000 dilution. Protein signals on the blot were quantified with the ImageJ program (NIH), and protein expression was normalized to control (100%). Numbers were determined using ImageQuant software in a nonsaturation range and after subtracting the background signal.