Mouse monoclonal anti actin antibody

L-929 cells incubated for 48 h in normoxia or hypoxia were solubilized in ice-cold RIPA buffer (1% Triton X-100; 1% deoxycholate, 150 mM NaCl, pH 7.2) containing inhibitors of proteases (Roche Applied Science, Mannheim, Germany) for 30 min on ice. Cell lysates were collected, cleared by centrifugation at 10,000× g for 10 min at 4 °C. Protein concentration was determined using the BCA protein assay reagent (Pierce, Rockford, IL, USA). Total protein extracts were separated in 10% SDS-PAGE and transferred onto PVDF membrane (Immobilon^TM-P, Milliopore, Billerica, MA, USA). For mCA IX detection, the membrane was incubated with mouse monoclonal AM4-3 antibody (10 μg/mL) diluted in blocking buffer for 2 h. Secondary anti-mouse peroxidase-conjugated antibody (Sigma–Aldrich, St. Louis, MI, USA) was diluted 1:5000 in blocking buffer. For CD63 analysis, mouse monoclonal antibody (1 μg/mL, ThermoFisher Scientific, Waltham, MA, USA) was diluted in 3% BSA in PBS with NP-40. For loading control, the membrane was probed with mouse monoclonal anti-actin antibody (Cell Signaling, Danvers, MA, USA). After treatment, the membrane was washed and developed by enhanced chemiluminescence using an ECL kit (Amersham Pharmacia Biotech, Buckinghamshire, UK).