2.6. Western Blot

Conditioned media was collected in sterile Eppendorf tubes and stored at −20 °C until further use. Cell lysates were isolated using 1× radioimmunoprecipitation assay (RIPA) buffer with a protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO, USA) and incubated on ice for 30 min, followed by centrifugation at 12,000× g for 15 min at 4 °C to pellet cell debris. The clear supernatant was isolated and stored at −20 °C until further use. A bicinchoninic acid (BCA) assay (ThermoScientific, Rockford, IL, USA) was performed to determine total protein levels followed by Western blot analysis with at least 90 μg protein loaded into each well. Sodium dodecyl–sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using Tris-glycine gradient gels (4–20%) (Novex, Life technologies, Carlsbad, CA, USA) electrophoresed at 130 V for 1.5 h followed by transfer onto a nitrocellulose membrane (0.45 μm) (BioRad, Hercules, CA, USA) at 100 V for 1 h on ice. A Ponceau stain (Figure S3) was performed following transfer to show equal protein loading by incubating Ponceau S solution (0.1% (w/v) in 5% acetic acid, Sigma Aldrich, St. Louis, MO, USA) with membrane for 5 min, followed by washing with distilled water for 2–3 min. Blots were blocked in 5% dry milk or 5% bovine serum albumin (BSA, BP1605-100, Fisher, Fair Lawn, NJ, USA) for 1 h at room temperature with shaking. The following primary antibodies were prepared at a 1:1000 dilution in 1–2% BSA in Tris-buffered saline with 0.1% Tween 20 immediately prior to use: collagen type I (ab34710), collagen type III (ab7778), collagen type V (ab94673), fibronectin (ab2413), and GAPDH (ab9485) (Abcam, Cambridge, MA, USA). The fluorescent secondary (Alexa Fluor 568, donkey anti-rabbit, Life Technologies, Eugene, OR, USA) (1:2000) was incubated with the probed membrane for 1 h at room temperature with rocking followed by washing and imaging (UVP Gel Imaging System, Upland, CA, USA).