C300 analyzer

Cells were lysed with NP-40 lysis buffer (1% Nonidet P-40, 0.05% sodium dodecyl sulfate, 50 mM Tris-Cl [pH 7.5], 150 mM NaCl, 0.5 mM sodium vanadate, 100 mM sodium fluoride, 50 mM β-glycerophosphate) supplemented with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Homogenates were centrifuged at 12,000× g for 15 min at 4 °C, and supernatants were collected. The protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride or nitrocellulose membranes. The membranes were blocked for 1 h at room temperature with 5% skim milk in Tris buffered saline-Tween buffer (0.1% Tween 20, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl). Membranes were incubated with indicated primary antibodies (anti-β-actin [DSHB, Iowa city, IA, USA], anti-Col1A1 [SouthernBiotech, Birmingham, AL, USA], anti-α-SMA [Abcam, Cambridge, CB2 0AX, UK]) at 4 °C overnight and then with horseradish peroxidase-conjugated secondary antibody. The chemiluminescence signal was detected with an Azure Biosystems C300 Analyzer (Azure Biosystems, Dublin, CA, USA) using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA).

Lysates of cells and harvested liver tissues were prepared using RIPA buffer (Thermo Scientific, Waltham, MA, USA) containing protease inhibitor (Sigma-Aldrich), phosphatase inhibitor cocktail 2 (Sigma-Aldrich), and phosphatase inhibitor cocktail 3 (Sigma-Aldrich). Total protein concentration of the lysates was measured using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Proteins were resolved by SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA), and probed with appropriate dilutions of the following antibodies: p53 (sc-6243, 1:1000, Santa Cruz), p21 (ab109199, 1:1000, Abcam, Cambridge, MA, USA), PAI-1 (sc-5297, 1:1000, Santa Cruz), TTP (T5327, 1:2000, Sigma-Aldrich), and α-tubulin (2125S, 1:1000, cell signaling, Danvers, MA, USA). Then, membranes were incubated with secondary antibodies (115-035-003; HRP-Goat Anti-Mouse IgG, 111-035-003; HRP-Goat Anti-Rabbit IgG) at room temperature for 30 min. Antibody binding was visualized with an ECL chemiluminescence system (Pierce Biotechnology) and chemiluminescence signal was read by Azure Biosystems C300 analyzer (Azure Biosystems, Dublin, CA). The relative band density was analyzed by using ImageJ2x software (US National Institutes of Health, Bethesda, USA).