Western Blot Analysis of Fibrosis Markers

Western blot was performed as previously described [19 (link)]. Membranes were incubated with anti-p-Smad3 (1:1,000; Cell Signaling Technology, Danvers, MA, USA), anti-Smad3 (1:1,000; Cell Signaling Technology), anti-PAI-1 (1:1,000; BD Biosciences), anti-α-SMA (1:1,000; Sigma), and anti-type I collagen (1:1,000; Abcam) polyclonal antibodies at 4℃ with gentle shaking overnight. Antibodies were detected by horseradish peroxidase-linked secondary antibody (Santa Cruz) using an Enhanced Chemiluminescence Western Blotting Detection System, in accordance with the manufacturer's instructions (Millipore, Billerica, MA, USA) [19 (link)]. The membrane was reblotted with anti-β-tubulin antibody (Applied Biological Materials Inc., Richmond, BC, Canada) to verify equal protein loading in each lane. Densitometric measurements of the bands were performed using UN-SCAN-IT digitizing software (Silk Scientific Corp., Orem, UT, USA).