The flow Ixazomib supplier probe and the two pressure transducers were connected to a PowerLab (4SP) linked to a computer using the Chart version 5.0.1 for Windows software (ADInstruments, Mountain View, CA). The average portal flow, inflow, and outflow pressures were continuously sampled, recorded, and afterward blindly analyzed under code. The perfused rat liver preparation was allowed to stabilize for 20 minutes before the studied substances were added. A normal gross appearance of the liver and

a stable perfusion pressure and perfusate pH (7.4 ± 0.1) were required during this period. If any viability criterion was not satisfied, the experiment was discarded. Sinusoidal endothelial function was explored by testing the vasodilation of the liver circulation to increasing concentrations of acetylcholine (10−7, 10−6, 10−5 M) added to the system, after preconstruction with the alpha-adrenergic agonist methoxamin (10−4 M). At the end of the vascular study liver samples were obtained and immediately frozen in liquid nitrogen and kept at −80°C until processed as described.24 Aliquots from each sample containing equal amounts of protein (40-100 μg) were run on an 8%-15% sodium dodecyl sulfate

(SDS)-polyacrylamide gel and transferred to a nitrocellulose membrane. Equal loading was ensured by Ponceau staining. The blots were subsequently blocked for 1 hour with Tris-buffered saline and probed overnight at 4°C with a mouse antibody recognizing endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) (BD Transduction Laboratories, Lexington, KY), AZD9668 a rabbit antibody recognizing phosphorylated eNOS at Ser1176 (BD Transduction Laboratories), a mouse antibody for nitrotyrosine (Cayman Chemical Co.), a rat antibody recognizing ICAM-1 (R&D Systems), a 3-mercaptopyruvate sulfurtransferase mouse antibody for TLR-4 (Toll-like receptor 4; Santa Cruz Biotechnology, Santa Cruz, CA), and a rabbit antibody

recognizing activated casapse-3 (Cell Signaling Technology). This was followed by incubation with rabbit antimouse (1:10,000) or goat antirabbit (1:10,000) horseradish peroxidase (HRP)-conjugated secondary antibodies (Stressgen, Victoria, BC, Canada) for 1 hour at room temperature. Blots were revealed by chemiluminescence and digital images were taken by a luminescent image analyzer LAS-4000 (General Electric, Little Chalfont, Buckinghamshire, UK). Protein expression was determined by densitometric analysis using the Science Lab 2001, Multi Gauge V2.1 (Fuji Photo Film, Düsseldorf, Germany). Quantitative densitometry values of iNOS, nitrotyrosine, ICAM-1, and caspase-3 were normalized to glyseraldehyde-3-phosphate dehydrogenase (GAPDH) and displayed in histograms. The degree of eNOS phosphorylation at Ser1176 was calculated as the ratio between the densitometry readings of P-eNOS and eNOS blots.