Enhanced chemiluminescence reagent

Cell lysates were prepared using lysis buffer (1% SDS, 1 mM Na-vanadate, 10 mM Tris-HCl pH 7.4) in the presence of 0.1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail. Proteins (40 μg) were subjected to SDS-PAGE and electroblotting on nitrocellulose membranes, which were probed with specific primary and HRP-conjugated secondary antibodies. Immunoblots were revealed by enhanced chemiluminescence reagent (Bio-Rad). Images were acquired using the VersaDoc 1000 imaging system and individual band densities were integrated by Quantity One software (BioRad).

RAW264.7 macrophages were collected and lysed in ice-cold radioimmunoprecipitation assay buffer (Cell signaling, MA, USA) for 30 min. The protein contents were measured by Bradford protein assay (Bio-Rad, CA, USA). Total proteins (20 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro-transferred onto polyvinylidene difluoride membrane (Millipore, Darmstadt, Germany). The membranes were blocked with 5% nonfat-milk for 30 min at room temperature and incubated in a 1:1000 dilution of the primary antibody overnight at 4 °C. The primary antibodies used were phospho Nf-κB p-65 (p-p65), phospho-IκB-α (p- IκB-α), Cox-2, iNOS, and β-actin. All antibodies were purchased from Cell Signaling (USA). The membranes were washed three times with Tris-buffered saline plus Tween 20 for 10 min each time. After that, the membranes were incubated in a 1:2000 dilution of horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The protein reactions were detected using enhanced chemiluminescence reagent (Bio-Rad, CA, USA) and chemi-luminator (Davinch-K, Seoul, Korea).

Cellular lysates were prepared from liver tissue, RAW264.7 and AML12 cells, respectively, by homogenizing tissues or cells in the lysis buffer (AR0101/0103; Boster Biological Technology Co. Ltd.). According to the manufacturer's protocol, the Pierce BCA protein assay kit (P0010; Beyotime) was used to detect protein concentration. After heat denaturation at 100°C for 10 min, 50 μg of liver tissue and cell lysates were into SDS‐PAGE and separated by electrophoresis, and then proteins were transferred to polyvinylidene fluoride membranes (Millipore). The membranes were first blocked with 5% skim milk and then incubated with the primary antibodies against MARCO, TLR4, TRIF, TNF‐a, IkB‐a, NF‐kB, Lamin B and GADPH, respectively at 4°C overnight or at room temperature for 2 h followed by incubation of the secondary antibodies conjugated with horseradish peroxidase at room temperature for 1–1.5 h. The bands were displayed with enhanced chemiluminescence reagent (Bio‐Rad). Then the protein band density was analysed by Image J or Image Lab analysis software and normalized to their respective controls.

Total cell lysates were generated with RIPA buffer (Sigma) containing proteinase and phosphatase inhibitors (Roche). Protein concentrations were measured using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific). Equal protein quantities were separated by SDS-PAGE (10% polyacrylamide), transferred to polyvinylidene difluoride membranes, incubated with primary and secondary antibodies, and visualized with an enhanced chemiluminescence reagent (Bio-Rad).