Western blot

Total protein was extracted from specimens using radioimmunoprecipitation assay (RIPA) buffer containing phenylmethylsulfonyl fluoride (PMSF). Proteins were denatured at 95 °C after concentration measurement, then 30 μg of the total protein was separated from these samples by 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) then transferred onto polyvinylidene fluoride (PVDF) membranes. After being blocked, membranes were sequential blotted with primary and secondary antibodies (1:10000). Signals were detected with an Odyssey infrared imaging system (LI-COR Bio, USA). Primary antibodies were as follows: anti-Nrf2 (1:500), anti-GPx1 (1:500), anti-MnSOD (1:500), anti-TGF-β1 (1:250), anti-Smad2 (1:250), anti-p-Smad2 (1:250), anti-Smad3 (1:250), anti-p-Smad3 (1:250), anticollagen I (1:100), anticollagen III (1:250), anti-α-SMA (1:5000), and anti-tissue inhibitor of metalloprotease (anti-TIMP-3) (1:1000).