Peroxidase labeled sheep anti mouse igg

The cells were harvested using lysis buffer (50 mM NaCl, 150 mM Tris-HCl, pH 7.4, 1% Nonidet P-40) with protease inhibitors (Halt protease inhibitor cocktail, Thermo Scientific), and proteins were extracted by rotation of the cell lysates for 30 min at 4°C. The cell lysates were then centrifuged on a high-speed tabletop centrifuge for 15 min, and the supernatants were collected to perform western blotting. CFTR was detected with IgG2b 596 (596 obtained from Dr. Jack Riordan at the U. of North Carolina at Chapel Hill), diluted 1:1000. STX8 was detected using a mouse monoclonal anti-STX8 antibody (BD Transduction Laboratories 611352). GAPDH protein was used as the loading control and detected with mouse monoclonal anti-GAPDH antibody (Santa Cruz Biotechnology). Peroxidase-labeled sheep anti-mouse IgG (Amersham) was used as the secondaiy antibody. The signal was enhanced with SuperSignal West Dura Extended Duration Substrate (Thermo Scientific). Chemoluminescence was captured by a Fuji Film LAS-4000 plus system with a cooled CCD camera. Quantification was carried out within the linear range, using Image Gauge version 3.2 software (Fuji Film).