Protein Extraction and Western Blot Analysis

Cultured cells were lysed in RIPA buffer (Beyotime Inc., NanTong, China). Protein concentration was identified using the Bradford reagent (Beyotime Inc.). We then performed electrophoresis of protein extracts and subsequent blotting as previously described [91 (link), 92 (link)]. In brief, Equivalent amounts of protein (30 μg) were separated by SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). The membranes were then immunoblotted with the appropriate primary antibodies against Smad3 or p-Smad3 (Santa Cruz Biotechnology, Santa Cruz, CA), at 4°C for overnight, and subsequently were incubated with HRP conjugated anti-mouse or anti-rabbit secondary antibodies at room temperature. Signals were detected using an enhanced chemiluminescence (ECL) system (Beyotime, China) on Kodak X-ray film. Equal protein loading was assessed by the expression of β-actin. The protein bands were quantified using the BioRad Quantity One software package.