In separate experiments, treated cells were lysed with either 20 mM Tris buffer (pH 7.5) containing 150 mM NaCl, 1% Triton X-100, and 1 mM EDTA supplemented with a cocktail of protease (Roche Diagnostics Corp., Indianapolis, IN, USA) and phosphatase (Sigma-Aldrich) inhibitors and subsequently clarified by centrifugation (20,000g × 10 minutes at 4°C) for phospho- and total-Smad protein analyses or with 2 times Laemmli sample buffer. To analyze secreted proteins, equal aliquots of conditioned media were collected and concentrated by centrifugal filtration (Amicon ultra centrifugal filters, 10-kDa cutoff; MilliporeSigma, Burlington, MA, USA). Clarified cell lysates and concentrated conditioned media were stored at -80°C until use. Protein concentrations in cell lysates and in culture media concentrates were determined by the BCA method (ThermoFisher Scientific) using BSA as a standard. Proteins (10 µg proteins per lane) in cell lysates or concentrated media were resolved by 4% to 20% SDS-PAGE gel electrophoresis either under reducing/denaturing (for Smads, collagens IV) or reducing (collagen I) conditions and electrotransferred overnight onto 0.2 µm nitrocellulose membranes. Membranes were blocked with 5% Carnation non-fat dried milk (Nestle, Arlington, VA, USA) in 10 mM Tris buffer (pH 7.4) containing 0.1% Tween 20 for 1 hour at 23°C, incubated overnight at 4°C in the presence of a 1:1000 dilution of rabbit monoclonal: (i) anti-Smad2 (clone 86F7), (ii) anti-phospho-Smad2 (clone 138D4; Ser465/467), (iii) anti-Smad3 (clone C67H9), or (iv) anti-phospho-Smad3 (clone C25A9; Ser423/425) primary antibody from Cell Signaling Technology. To quantify relative changes in Nox4 protein content, a 1:2000 dilution of rabbit monoclonal anti-NADPH oxidase 4 (ab133303; Abcam, Cambridge, MA, USA) primary antibody was used, as indicated. Relative changes in collagens were determined using a 1:1000 dilution of sheep polyclonal anti-collagen 1α1 (AF6220-SP; R&D Systems Inc., Minneapolis, MN, USA) or a 1:1000 dilution of rabbit polyclonal anti-collagen IV (ab6586; Abcam) primary antibodies were used as indicated. Immunostained membranes were washed in Tris-Tween buffer and incubated for 1 hour at 23°C with a 1:10,000 dilution of goat anti-rabbit IgG (Jackson ImmunoResearch Labs, Inc, West Grove, PA, USA) or a 1:1000 dilution of goat anti-sheep IgG (R&D Systems, Inc.) horseradish peroxidase-conjugated secondary antibodies, respectively. Immunostained proteins were visualized using Supersignal West Pico chemiluminescent substrate (ThermoFisher Scientific). Relative changes in protein content were quantified by densitometry and normalized to total Smad or total GAPDH content, as indicated.