Western Blot Analysis of Fibrosis Markers

Anti-alpha SMA. (A2547; Sigma-Aldrich), anti-collagen (#90395; Abcam, Cambridge, MA), anti-TGF-β (#66043; Abcam), anti-PDGFR-β (sc-432; Santa Cruz Biotechnology, Santa Cruz, CA), anti-GAPDH (sc-1694; Santa Cruz Biotechnology), anti-p-MEK1/2 (#2338; Cell Signaling Technology, Danvers, MA), anti-MEK1/2 (#8727; Cell Signaling Technology), anti-p-ERK1/2(p44/42) (#4370; Cell Signaling Technology), anti-ERK (#4695; Cell Signaling Technology), and p-CREB (#9198; Cell Signaling Technology) were used for western blotting. Cell or tissues were lysed with RIPA buffer (G-Biosciences, St. Louis, MO, USA) with a protease inhibitor cocktail followed by western blotting as previously described^{41 (link)}. The protein concentration of the lysates was quantified by using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA) Denatured proteins were loaded into SDS–polyacrylamide gel, resolved by electrophoresis, and transferred to a nitrocellulose membrane, blocked by incubation with 3% bovine serum albumin (Sigma) for 1 h at room temperature, and the membrane was then incubated with each primary antibody overnight. Incubation with the appropriate secondary antibodies conjugated to HRP was performed for 1 h. Protein bands were developed onto an X-ray film (Fujifilm) after incubation with enhanced chemiluminescence.