Image gauge version 3

Protein samples were electrophoresed on SDS/PAGE (7.5 % gels] and transferred to PVDF membranes. Membranes were blocked with 5% non-fat dried skimmed milk in TBST Tris-buffered saline (TBST: 50 mM Tris and 150 mM NaCl [pH 8.0] with 0.05% Tween 20] for 1 h, then incubated overnight at 4°C with an anti-ABCA4 antibody (sc-65672, Santa Cruz Biotechnology). Ezrin (sc-58758, Santa Cruz Biotechnology) detection was used as a loading control. The blots were then washed and incubated with sheep anti-mouse IgG peroxidase-linked antibody (GE Healthcare). Immunoreactive bands were visualized by SuperSignal West Dura substrate (Thermo Scientific) using a Fuji Film LAS-4000 Plus system with a cooled CCD camera. Image Gauge version 3.2 software (Fuji Film) was used for quantification of the bands. Background was determined in the vicinity of each of the band quantified and the background values subtracted. For experiments involving lysate samples, 80–100 ng of protein was loaded into the gel. For surface biotinylation, 3000–3500 μg was loaded.

The cells were harvested and solubilized in lysis buffer (described above), supplemented with protease inhibitor cocktail (catalogue 78429, Thermo Scientific). The cell lysates were centrifuged at 14000g for 20 min at 4°C to pellet the insoluble material. The supernatants (50 μg of proteins) were subjected to 10% SDS-PAGE and immunoblotting, followed by enhanced chemiluminescence (SuperSignal West Dura Extended Duration Substrate, catalogue 34075, Thermo Scientific). The chemiluminescent signal on the polyvinylidene difluoride (PVDF) membrane (Bio-Rad) was directly captured by a FujiFilm LAS-4000 Plus system with a cooled CCD camera. CFTR protein was detected with monoclonal anti-human CFTR (217; 1:1,000) antibody (provided by Dr. John Riordan, University of North Carolina). Ezrin, used as a loading control, was detected with monoclonal antibody (1:10,000; Santa Cruz Biotechnology). Mouse monoclonal antibodies directed against VCP (1:500), HDAC7 (1:1000), AHSA1 (1:1000), Hsp90 (1:1000), Hsp70 (1:1000), Hsp40 (1:1000), Hsp27 (1:500) and a rabbit polyclonal antibody specific for HDAC6 (1:500) were used. Image Gauge version 3.2 software (Fuji Film) was used to quantify the bands.