Vx 661

Stocks of aminoglycoside antibiotics (All purchased from Sigma-Aldrich, St. Louis, MO, USA) were dissolved in sterile H₂O at a 100 mg/mL concentration, and stored at 4 °C. PTC124 (3-[5-(2-Fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid, MedChemExpress, Cat. #HY-14832), escin (Sigma-Aldrich, Cat. #E1378), VX-661 (MedChemExpress, Monmouth Junction, NJ, USA Cat. #HY-15448), NMDI-14 (Ethyl 2-(((6,7-dimethyl-3-oxo-1,2,3,4-tetrahydro-2-quinoxalinyl)acetyl)amino)-4,5-dimethyl-3-thiophenecarboxylate, EMD Millipore, Burlington, MA, USA, Cat. #530838), and amlexanox (MedChemExpress Cat. #HY-B0713) were dissolved in dimethylsulphoxide (DMSO). SMG1i (2-chloro-N,N-diethyl-5-((4-(2-(4-(3-methylureido)phenyl)pyridin-4-yl)pyrimidin-2-yl)amino)benzenesulfonamide) was received from Dr. Robert Bridges from Rosalind Franklin University of Medicine and Science through the CFFT compound distribution program, and was dissolved in DMSO. Working solutions were all ≤0.1% DMSO, with the exception of NMDI-14, which was 1% DMSO. The increased DMSO did not prevent G418-facilitated FIS (Supplementary Figure S9). Stocks of forskolin (Sigma-Aldrich, Cat. #F6886) were dissolved in 100% EtOH and stored at −20 °C.

CF HBE cells were infected with adenoviruses (wt-CFTR, F508del-CFTR or S13F-CFTR, 50 MOI), cultured to ∼90% confluency, and exposed to the Trikafta correctors VX-445 (3 μM, MedChemExpress) and VX-661 (3 μM, MedChemExpress), or maintained at low temperature (29°C) for 24 h (in OptiMEM). Cells were rinsed twice with ice cold PBS and lysed [150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl pH 7.4, 0.5% Triton X-100 and one tablet of protease inhibitor cocktail (Roche)]. Lysates were centrifuged (16,200 g for 20 min) at 4°C and total protein content was measured using Pierce™ BCA protein assay (Thermo Scientific). Aliquots containing 700–1000 μg protein were precleared with 20 μl Protein G Sepharose™ 4 Fast Flow beads for 30 min at 4°C then incubated with the rabbit anti-FLNA antibody (1:500 dilution, Invitrogen) for 30 min at 4°C. Immunocomplexes were precipitated using Protein G Sepharose beads, washed five times with lysis buffer, eluted using 2× Laemmli buffer, and analyzed by immunoblotting. The CFTR monoclonal antibody 596 (1:1000, CFFT clone#A4) was used, and was validated in Baby Hamster Kidney (BHK) cells by immunoblotting lysates of the cells with or without transfection with wt-CFTR.