AEC were lysed on ice in RIPA (Radioimmunoprecipitation assay) buffer (50 mM Tris pH 7,4, 150 mM NaCl, 1% (v/v) Igepal, 1% (w/v) sodium deoxycholate, 5 mM iodoacetamide and 0,1% (v/v) SDS) supplemented with protease inhibitor cocktail inhibitor (Roche diagnostics), then lysates were clarified by centrifugation (13,000 × g for 10 min at 4 °C). Ten µg of total proteins quantified by Biorad DC protein Assay Reagent Package (Biorad) were submitted to electrophoresis. Subcellular Protein Fractionation Kit (Thermo Scientific) was used to resolve proteins according to their subcellular localizations following the manufacturer's instructions.
Primary antibodies were anti–Gli1 (AF3455, R&D Systems), anti-Gli2 (HPA074275, Sigma Aldrich), anti-Gli3 (HPA005534, Sigma Aldrich), anti-Patched1 (E-AB-10571, Elabscience), anti-Smoothened (NBP2-24543, Novus Biologicals), anti-Ck5 (AB24647, Abcam, Cambridge), anti-GAPDH (clone 6C5, Chemicon, Millipore), anti-Foxj1 (14–9965–82, Ebioscience, ThermoFisher Scientific). Membranes were incubated with appropriate dilutions of primary antibody followed by the appropriate peroxidase-conjugated secondary antibody (Bio-Rad Laboratories). Final detection was obtained by enhanced chemiluminescence (GE Healthcare). Detected signals were digitally compared using ImageJ.
Primary antibodies were anti–Gli1 (AF3455, R&D Systems), anti-Gli2 (HPA074275, Sigma Aldrich), anti-Gli3 (HPA005534, Sigma Aldrich), anti-Patched1 (E-AB-10571, Elabscience), anti-Smoothened (NBP2-24543, Novus Biologicals), anti-Ck5 (AB24647, Abcam, Cambridge), anti-GAPDH (clone 6C5, Chemicon, Millipore), anti-Foxj1 (14–9965–82, Ebioscience, ThermoFisher Scientific). Membranes were incubated with appropriate dilutions of primary antibody followed by the appropriate peroxidase-conjugated secondary antibody (Bio-Rad Laboratories). Final detection was obtained by enhanced chemiluminescence (GE Healthcare). Detected signals were digitally compared using ImageJ.