Hybond c nitrocellulose membranes

Proteins (30 μg) separated by 10% SDS-PAGE and 3–8% SDS-PAGE (0.1% SDS) were transferred to Hybond-C nitrocellulose membranes (Life Technologies, Carlsbad, CA, USA by electroblotting. The blot was incubated with a mouse antibody anti–CFTR (Ab570 1:500), and HRP-conjugated anti-mouse (PIERCE, 1:5000). The target protein was detected by ECL reagents (Pierce -Thermo Fisher Scientific, Rodano, Italy)). Anti-β-tubulin antibody (mouse; Sigma-Aldrich, St. Louis, MO, USA, 1:10,000) confirmed the presence of an equal amount of proteins per well. Gel bands were quantified by ImageJ software.

Proteins were extracted using NucleoSpin RNA/protein (Macherey-Nagel) and quantified by the Bicinchoninic acid method. For each experimental condition, 30 μg of proteins were separated by electrophoresis on Bis-Tris gel (GenScript Biothec, Leiden, Netherlands) and transferred onto Hybond-C-nitrocellulose membranes (Life Technologies Ltd. Paisley, UK).
After 1 h in blocking solution (SuperBlock Blocking buffer in PBS, Thermo Scientific), membranes were incubated overnight at 4 °C with the following primary antibodies: anti-human αSMA (dilution 1:300, Sigma-Aldrich), S100A4 (dilution 1:100, Santa Cruz Biotechnology, Dallas, USA), COL1 (dilution 1:600, Proteintech), and FN (dilution 1:2,000, Sigma-Aldrich). The membranes were subsequently incubated with (HRP)-conjugated secondary antibodies (dilution 1:2,000; Cell Signaling, MA, USA). To confirm similar loading of protein samples into the gels and the efficiency in the electrophoretic transfer, membranes were incubated with primary HRP-conjugated antibody to human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (dilution 1:2,000; Santa Cruz Biotechnology). Protein synthesis was detected using the enhanced chemiluminescence system (Luminata Crescendo, Millipore), and the densitometric analysis was performed by the UVITEC Image Analysis System (UVITEC, Cambridge, UK).