Bicinchoninic acid assay reagent

Protein levels in liver tissues were extracted using RIPA buffer (150 mM NaCl, 50 mM Tris–HCl pH 7.5, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, and 1 mM EDTA)^{157 (link)} containing proteinase inhibitor cocktail (ML051, HIMEDIA, India). Cell lysates were collected, and the concentration was quantified by bicinchoninic acid assay reagent (Thermo Fisher Scientific, USA). By electrophoresis on a sodium dodecyl sulfate polyacrylamide gel, equal amounts of proteins were separated and transferred to a polyvinylidene fluoride membrane. Then, the membranes were incubated with OneStep Blocker solution (BS001, Genedirex, USA), followed by anti-cleaved caspase-3 (Thermo Fisher Scientific, USA), IL-6 (Abcam, USA), TNF-α (Merck, Germany), TNF-β1 (Abcam, USA), and α-SMA (Cell Signaling, USA) primary antibodies and then exposed to horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Cell Signaling, USA) or goat anti-mouse secondary antibody (Invitrogen, USA). β-actin (Cell Signaling, USA) was used as an internal control. Finally, protein bands were visualized using Luminata TM Forte Western HRP Substrate (Merck, Germany) and detected by chemiluminescence western blot detection (Image Quant LAS 4000; GE Healthcare Life Science, USA). Percentages of relative expression levels of protein/actin were calculated by ImageJ software version 1.46.

Protein sample buffer and bicinchoninic acid assay reagent were from Thermo Scientific (Rockford, IL). Closantel was from Sigma (St. Louis, MO) and WNK463 was from MedChemExpress (Princeton, NJ). STOCK1S-50699 and STOCK1S-14279 were from InterBioScreen (Chernogolovka, Russian Federation). Horseradish peroxidase (HRP)-conjugated anti-rabbit Ig was from Molecular Probes (Eugene, OR). RIPA buffer and enhanced chemiluminescence agent (ECL) reagent were from Pierce (Rockford, IL).

The cells were cultured to 70% confluency and incubated in serum-free media containing various concentrations of ursolic acid (0.0, 1.0, 2.5 and 5.0 μM) for 48 h. The conditioned medium was collected and centrifuged at 13,000 × g for 10 min to remove cell debris. Subsequently, the protein concentration was measured using bicinchoninic acid assay reagents (Pierce Biotechnology, Inc., Rockford, IL, USA), and equal amounts of protein from the conditioned media were electrophoresed on 10% SDS-PAGE gels containing 1 mg/ml gelatin. Following electrophoresis, the gels were washed with renaturation buffer (2.5% Triton X-100) three times for 30 min, rinsed for 15 min with developing buffer [50 mM Tris-HCl buffer (pH 7.6) containing 5 mM CaCl₂, 0.02% Brij-35 and 0.2% sodium azide], and incubated overnight at 37°C. The gels were stained with staining buffer (0.5% Coomassie Brilliant Blue R-250 solution containing 10% acetic acid and 20% methanol) for 30 min and destained with 10% acetic acid solution. Areas of gelatinase activity were detected as clear bands against the blue-stained gelatin background, and relative band intensities were determined by the quantification of each band using the Gel Doc™ XR+ imaging system (Bio-Rad Laboratories).