Protein isolation and Western blot analysis
Cells were harvested 48 hours post-transfection, washed twice in 1x phosphate-buffered saline (PBS), and lysed in boiling buffer (1% [wt/vol] sodium dodecyl sulphate, 1 mM Na3VO4, 10 mM Tris, pH 7.4) containing 0.1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 5 μg/ml leupeptin for 10 min at 4°C. Before separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, 40 µg of proteins (20µl volume) were boiled in Laemmli buffer for 5 min. Proteins were then electroblotted onto polyvinyl difluoride membranes (Bio-Rad, Ivry-sur-Seine, France), and nonspecific binding sites were blocked for 2h at room temperature by 6% (w/v) fat-free milk before an overnight incubation at 4°C with specific antibodies for CFTR 570 (1:1000, CFTR Folding Consortium, University of North Carolina,USA) and Tubulin (1:5000). Primary antibodies were detected with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgGs (1:10,000; Jackson ImmunoResearch Laboratories, Villepinte, France) or rabbit anti-goat IgGs (1:5,000; Dako, Glosturp, Denmark). Blots were revealed using an Enhanced Chemioluminescence detection kit (Amersham, Les Ulis, France).