Chemidoc it 500 imaging system

Western blotting was performed as described previously^{31 (link)}. Tissue samples were lysed in buffer containing 0.1% Triton X100 and a cocktail of protease inhibitors (Sigma-Aldrich). Protein concentration was measured by Bradford assay (Bio-Rad Laboratories). Protein extracts were then separated on 8–12% SDS-PAGE and transferred onto PVDF membrane (GE Healthcare Life Sciences). Membranes were probed with primary antibodies against p16^INK4 (ab54210), SIRT1 (ab110304), acetyl-p53^Lys381 (ab61241), TGF-β (ab66043), SMAD3 (ab28379), IL-6 (ab9324), TNF-α (ab6671), MMP2 (ab86607) (Abcam); p53 (sc126, Santa Cruz); Bcl-2 (B9804, Sigma-Aldrich); phospho-SMAD3^Ser423/425 (9,520), SMAD2/3 (8685S, Cell Signaling Technology); NF-κB p65 (PA5-16,545), acetyl-SMAD2/3 (PA576015, ThermoFisher). Loading conditions were determined with GAPDH (G8795, Sigma-Aldrich). Images were obtained by ChemiDoc-it 500 Imaging System (Bio-Rad Laboratories) and the optical density of the bands was analyzed with Quantity One Analysis Software (Bio-Rad Laboratories).

The liver was homogenised with a dunce homogeniser at 4 °C in ice-cold RIPA buffer (3 mL/g tissue, added with protease inhibitor cocktail, all from Santa Cruz Biotechnology). After the addition of 300 μg of Phenylmethylsulfonyl fluoride (PMSF) per gram of tissue, lysate was kept in ice for 30′ and then centrifuged twice at 10,000× g for 10 min at 4 °C to gain total proteins extract in cleared supernatant. Protein concentration was then determined using the Lowry method [77 (link)].
Proteins (40 µg) were resolved by 8 or 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred into PVDF filters by Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Inc. Hercules, CA, USA). Filters were then stripped in Western blot stripping buffer (Santa Cruz Biotechnology Inc.) and reprobed with actin-β to normalise protein signals. Images were obtained by ChemiDoc-it 500 Imaging System (Bio-Rad Laboratories), and the optical density of the bands was analysed with Quantity One Analysis Software (Bio-Rad Laboratories). Data were expressed as mean protein/α-actin ratio ± SD.