Western Blot Analysis of CFTR Variants

The CFBE cells were solubilized in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and pH 8.0) supplemented with 1 mM PMSF, 5 µg/mL leupeptin, and pepstatin. Western blotting experiments were performed as reported previously [43 (link)]. After detecting the total proteins in the blotting membrane by Ponceau S (Sigma-Aldrich, St. Louis, MO, USA) staining, the HBH-CFTR-3HA variants were detected by an anti-HA antibody (16B12, BioLegend, San Diego, CA, USA) and an HRP-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA). The antigen–antibody complexes were incubated with a SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher, Waltham, MA, USA) and analyzed by the Fusion chemiluminescence imaging system (Vilber Bio Imaging, France).