Mouse monoclonal antibodies to α tubulin

Myocardial tissues and H9C2 cells were homogenized in ice-cold RIPA buffer (25 mmol/L Tris·HCl [pH 7.6], 150 mmol/L NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail, and phosphatase inhibitor cocktail). The homogenate sample was sonicated briefly before centrifugation at 13,500 ×g for 30 min at 4℃. The BCA assay kit (Pierce, Rockford, IL, USA) was used to quantitate protein. Protein (50~100 µg) was separated by SDS-PAGE on a 10~12% polyacrylamide gel. Proteins were then transferred to nitrocellulose membranes (GE Healthcare, Chalfont St. Giles, UK) and blocked in 5% skim milk for 2 h at room temperature. Membranes were then probed with mouse or rabbit polyclonal antibodies to Dcn, Smad2, or pSmad2 (Abcam), or with mouse monoclonal antibodies to α-tubulin (Sigma-Aldrich) at a 1:500 dilution overnight at 4℃. Following three washes in PBS with Tween 20 (PBS-T, pH 7.4), membranes were incubated with horseradish peroxidase-conjugated antimouse or anti-rabbit IgG secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) at a 1:2000 dilution for 2 h at room temperature. Following washing with PBS-T three times, a western blotting detection kit (Abclon Inc., Seoul, Korea) was utilized to visualize the antibody-bound proteins. These experiments were performed in triplicate.