Ivacaftor vx 770

Ion channel activities from 16HBEge cells and fully differentiated CFBE cells were recorded in Ussing chambers as described previously [11 (link)]. Briefly, Snapwell or Transwell filters were mounted in Ussing chambers (EasyMount chamber; Physiologic Instruments, Reno, NV, USA) connected to a VCC MC8 voltage clamp unit (Physiologic Instruments, Reno, NV, USA). Solutions were maintained at 37 °C by heated water jackets and bubbled with CO₂. CFTR-dependent short-circuit currents (I_SC) were measured as the change in I_SC after CFTR inhibition by 10 µM CFTR_inh-172 (Cat. No. C2992; Sigma-Aldrich, St. Louis, MO, USA) in the presence of 10 µM amiloride (Cat. No. A7410; Sigma-Aldrich, St. Louis, MO, USA) following CFTR potentiation and stimulation by 1 µM ivacaftor (VX-770) and 10 µM forskolin (Cat. No. F3917; Sigma-Aldrich, St. Louis, MO, USA), respectively. CaCC was stimulated by 100 µM UTP (Cat. No. ab146222; Abcam, Cambridge, MA, USA).

RPMI 1640, fetal bovine serum (FBS), penicillin–streptomycin solution 100X, L-glutamine 100X 200 mM, Lympholyte^®-H, and prestained protein SHARPMASS VI MW marker were purchased from Euroclone SpA (Milan, Italy); anti-MMP9 antibody, horseradish peroxidase (HRP)-linked anti-rabbit secondary antibody, protease inhibitor cocktail (100X), and phosphatease inhibitor cocktail (100X) were obtained from Cell Signaling Technology (Danvers, MA, USA); dibutyryl-cAMP, isopropyl β-D-1-thiogalactopyranoside (IPTG), yeast extract, tryptone, PGex6P1, 6,7-Dihydro-7,9-dimethyl-6-(5-methyl-2-furanyl)-11-phenylpyrimido (4′,5′,3,4) pyrrolo (1,2-a) quinoxaline –8,10 (5H,9)-dione (PPQ-102), a reversible and voltage-independent CFTR inhibitor, and the potentiator Ivacaftor (VX770) were purchased from Sigma-Aldrich (Milan, Italy); GSH-sepharose™, ECL Select™ Western Blotting Detection Reagent, and Amersham ™ Protran^® Premium 0.45-μm nitrocellulose were obtained from GE Healthcare (Chicago, IL, USA); Monocytes Isolation Kit II was purchased from Miltenyi Biotec Srl (Bologna, Italy); pEYFP-C1 plasmid was obtained from Clontech Laboratories (Mountain View, CA, USA); QuickChange site-directed mutagenesis kit was from Stratagene (San Diego, CA, USA); BamHI and EcoRI restriction enzymes by Fermentas were purchased from Life Technologies Italia (Monza, Italy).