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).