Ab64085

Cells were lysed in RIPA buffer 1X (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) containing Complete Protease Inhibitor Cocktail (Roche, NJ, USA). The protein concentration in lysates was quantified using the Quantum Protein Assay kit (Euroclone). Total lysates were electrophoresed (30 μg per lane) with NuPAGE Novex Bis-Tris 4–12% gel (Life Technologies) and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) for Western blotting. The TMEM16A protein was immunodetected with the r [SP31] rabbit monoclonal antibody (ab64085, Abcam; 1:1000). The secondary antibody was anti-rabbit HRP (Millipore; 1:5000). Membranes were also stripped with the Restore Western Blot Stripping Buffer (Thermo Fisher Scientific Inc.) and incubated with the mouse monoclonal C464.8 (Merck Millipore) antibody against Na⁺/K⁺-ATPase β1 (1:6000) followed by anti-mouse HRP-conjugated secondary antibody (Ab 97023, Abcam; 1:10000). All antibodies were dissolved in 5% skimmed-milk in Tris-buffered saline—Tween 20. Protein bands were visualized using the ECL Advanced Western Blotting Detection Kit (GE Healthcare Europe, Little Chalfont, UK). Direct recording of the chemiluminescence was performed using the Molecular Imager ChemiDoc XRS System (Bio-Rad).

U251 cells growing on coverslips were transfected with Sc shRNA or 14-3-3 shRNAs and incubated for an additional 72 h. Cells were fixed in 4% PFA for 20 min at RT and then incubated with WGA647 conjugate (1:200; Thermo) at 4 °C for 15 min to preferentially label the plasma membrane. Cells were permeabilized with Triton X-100 (0.5% in PBS) and blocked with 5% BSA. Cells were incubated overnight at 4 °C with anti-ANO1 (1:100; ab64085, Abcam) or anti-14-3-3γ (1:100; ab118877, Abcam) antibodies. After washing, cells were incubated with DyLight 488- or 549-conjugated secondary antibodies (1:400; Jackson Labs) for 1 h at RT. Cells were washed, mounted, and observed under a Nikon A1 confocal microscope.

Protein was isolated from filter-grown BCi-NS1 cells using a lysis buffer containing 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 5% glycerol, 0.43% Nonidet P-40, 100 mM dithiothreitol (both from PanReac AppliChem, Barcelona, Spain) and 1× protease inhibitor mixture (Roche, Basel, Switzerland). Proteins were then separated by 8.5% SDS-PAGE and transferred to a PVDF membrane (GE Healthcare, Munich, Germany). Membranes were incubated overnight at 4 °C with rabbit primary antibodies against TMEM16A (#ab64085, Abcam, Cambridge, UK) and α-SLC26A9 (Davids Biotechnologie, Regensburg, Germany) and a mouse primary antibody against CFTR (#596, Cystic Fibrosis Foundation Therapeutics, Bethesda, MA, USA). Rabbit anti-actin (#A2066; Sigma-Aldrich, St. Louis, MO, USA) was used as a loading control. Membranes were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit/sheep anti-mouse secondary antibodies at room temperature for 2 h, and immunoreactive signals were visualized using a SuperSignal HRP chemiluminescence substrate-detection kit (#34577; Thermo Fisher Scientific, Waltham, MA, USA).