4.2. Antibodies and Reagents

The following primary mouse monoclonal antibodies were used, anti-CFTR (596) from Cystic Fibrosis Foundation Therapeutics (CFFT), Chapel Hill, NC [44]; anti-ezrin antibody 610603 from BD Biosciences (San Jose, CA, USA) anti-digoxigenin (DIG)-alkaline phosphatase antibody from Roche Applied Sciences (Indianapolis, IN, USA) and anti-Ago2 RN003M from Medical and Biological Laboratories (MBL) (Nagoya, Japan). The primary rabbit polyclonal antibodies, anti-TβR-I and anti-TβR-II (AP08190PU-N and AP54233PU-N, respectively) were from Acris (San Diego, CA, USA), anti-Smad2/3 5678 from Cell Signaling Technology, Inc. (Danvers, MA, USA). The horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit (BioRad Laboratories, Hercules, CA, USA) were used. All antibodies were used at the concentrations recommended by the manufacturer and previously validated.

Human TGF-β1 (Sigma-Aldrich, St. Louis, MO, USA), or vehicle control (4 mM HCl + 1 mg/mL BSA) was used at a previously established clinically relevant concentration (15 ng/mL) based on measurements in serum and the blood of CF patients (2.8 to 35 ng/mL) [22,62,63,84]. Pierce^TM Protease Inhibitor Mini tablets EDTA-Free (Thermo Scientific, Rockford, IL, USA) was used in lysis buffer in the RIP assay kit (MBL). Corrector VX-809 was from Selleck Chemicals (Houston, TX, USA), and the correctors C18 (VRT-534) and CFFT-002 were from CFFT. All correctors were used at 5 μM concentration to increase delivery of F508del-CFTR to the apical membrane in HBE cells. Correctors, or vehicle-control DMSO were added daily to basolateral medium of F508del HBE cells for 48 h [22]. The final concentration of DMSO was <0.1%. The CFTR_inh-172 (5 μM) was from Selleck Chemicals. Amiloride (50 μM), forskolin (20 μM), and IBMX (1 mM) were from Sigma-Aldrich.