Mm13 4

Cells were lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris- HCl, pH 8.0) supplemented with protease Inhibitor Complete Mini (Roche) on ice for 15 min. The cell lysates were rotated at 4 °C for 30 min and the insoluble material was removed by centrifugation at 15,000 g for 15 min. Protein concentrations were determined by BioRad™ Protein Assay using bovine serum albumin (BSA) as a standard. Following the normalization of protein concentrations, lysates were mixed with an equal volume of 2X Laemmli sample buffer and incubated for 5 min at 95 °C prior to separation by SDS PAGE on stain-free TGX gradient gels (BioRad). Following SDS-PAGE, the proteins were transferred to polyvinylidene fluoride membranes (300 mA for 90 min at 4 °C). The membranes were then blocked with BSA (Sigma-Aldrich) dissolved in PBS/Tween-20 (3% BSA, 0.5% Tween-20 for 1–2 h), followed by immunoblotting with the primary antibody specified for each experiment CFTR (Merck MM13–4 diluted at 1:1000), HIF-1α (Abcam ab16066, diluted at 1:1000); and beta ACTIN (Abcam ab1801, diluted at 1:1000). After the washing steps, the membranes were incubated with goat anti-rabbit IgG (H + L chains) or with goat anti-mouse IgG (H + L) HRP-conjugated secondary antibodies (BioRad) and detected using ECL (Amresco). Densitometry was performed using Image Lab software v. 4.1 (BioRad).

Nasal cultures were grown at 37°C for 48 h in the absence or presence of small molecules (3 μM VX‐809, 1 μM VX‐661, or 4 mM 4‐phenylbutyrate, 4‐PBA) as required. Cells were then lysed in modified radioimmunoprecipitation assay buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, pH 7.4, 0.2% (v/v) SDS, and 0.1% (v/v) Triton X‐100) containing a protease inhibitor cocktail (Roche) for 10 min, and the soluble fractions were analyzed by SDS–PAGE on 6% gels as described above and previously (Molinski et al, 2014; Pasyk et al, 2015). After electrophoresis, proteins were transferred to nitrocellulose membranes and incubated in 5% (w/v) milk, and CFTR bands were detected using the human CFTR‐MSD1‐specific (amino acids 25–36) murine mAb MM13‐4 (1:200, Abcam, Cambridge, UK), using horseradish peroxidase‐conjugated goat anti‐mouse IgG secondary antibody (1:2,500), and by exposure to film for 0.5 to 30 min as required. CNX was used as a loading control and detected using a CNX‐specific rabbit Ab (1:5,000, Sigma‐Aldrich), using horseradish peroxidase‐conjugated goat anti‐rabbit IgG secondary antibody (1:5,000), and by exposure to film for 0.5 to 5 min as required. Relative levels of CFTR proteins were quantitated by densitometry of immunoblots using ImageJ 1.42 Q software (National Institutes of Health), and reported values are normalized to CNX expression levels.