Total proteins were extracted in lysis buffer (150 mM NaCl, 20 Mm Tris-HCl, 2 mM EDTA, 1% Nonidet P-40, 0.1% SDS and inhibitor proteases) on ice for 1 h and then centrifuged for 15 min at 12,000×g and 4 °C. The supernatant containing total protein was recovered, and the protein concentration was evaluated using the Protein Assay Kit (Thermo Fisher Scientific, Strasbourg, France) and quantified by spectrophotometry at 450 nm.
Total protein extract (20 μg) was reduced and size-separated by 8% SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Marnes-la-Coquette, France), which were blocked in 5% BSA (PAA, Les Mureaux, France). Then, the membranes were incubated with specific primary rabbit polyclonal antibodies against ANO1 (ab53213, 1:10; Abcam, Paris, France) and mouse monoclonal β-actin (A5441, 1:500; Sigma, Saint Quentin Fallavier, France). The proteins of interest were detected using ECL detection system (Thermo Fisher Scientific). Relative quantification was performed by densitometric analysis using MultiGauge software (Fujifilm, Courbevoie, France). See Supplementary Fig.22 for gel source data.
Total protein extract (20 μg) was reduced and size-separated by 8% SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Marnes-la-Coquette, France), which were blocked in 5% BSA (PAA, Les Mureaux, France). Then, the membranes were incubated with specific primary rabbit polyclonal antibodies against ANO1 (ab53213, 1:10; Abcam, Paris, France) and mouse monoclonal β-actin (A5441, 1:500; Sigma, Saint Quentin Fallavier, France). The proteins of interest were detected using ECL detection system (Thermo Fisher Scientific). Relative quantification was performed by densitometric analysis using MultiGauge software (Fujifilm, Courbevoie, France). See Supplementary Fig.