Fluor s max multiimager system

The SDS-PAGE-separated proteins were transferred onto 0.45 μm pore-size PVDF membranes (Millipore; Burlington, MA, USA) using Trans-Blot (Bio-Rad; Hercules, CA, USA). The membranes were blocked in 5% zero-fat-dried milk in Tris-buffered saline containing 0.1% Tween 20 prior to the incubation in primary antibodies for 1–3 h at room temperature or overnight at 4 °C. The membranes were subjected to several washes in Tris-buffered saline containing 0.1% Tween 20 (TBST) to remove excess antibodies. The immunoreactive proteins were detected by incubation with a species-specific horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The immune complexes were detected with the Amersham Enhanced ChemiLuminescence system (Amersham Biosciences; Piscataway, NJ, USA) prior to scanning using the Fluor-S Max MultiImager system (Bio-Rad; Hercules, CA, USA).

The levels of TLR4, TNF-α, NF-κB p65, and phospho-NF-κB in liver tissue and hepatocytes were analyzed by western blotting. Proteins were extracted using RIPA buffer (Thermo, USA), according to the manufacturer’s instructions, and the protein concentrations were measured using a BCA protein assay kit (Thermo, USA). An equal amount of protein from each sample was subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). Membranes were blocked with 5 % milk in 1 × Tris-buffered saline with 0.1 % Tween-20 (TBST) for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies. Membranes were washed twice for 10 min in 1 × TBST and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h. Membranes were then washed twice for 10 min in 1 × TBST. Proteins were visualized by ECL (Millipore), and blots were scanned using a Fluor-S MAX MultiImager System (Bio-Rad). The signal intensities were determined using Quantity One image software (Bio-Rad).