Western Blot Analysis of TLR4 Expression

Western blotting was conducted as described previously (3 (link),12 (link)). Briefly, the membranes were blocked for 30 min at room temperature with 5% non-fat dry milk (Sigma-Aldrich, St. Louis, MO, USA) and immunoblotted using anti-TLR4 (cat. no. sc-293072; monoclonal mouse anti-rat; 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C. Following several washes, the membranes were incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated polyclonal goat anti-mouse IgG (cat. no. sc-2005; 1:2,000; Santa Cruz Biotechnology, Inc.) to detect TLR4 expression. Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; cat. no. sc-47724; monoclonal mouse anti-rat; 1:1,000; Santa Cruz Biotechnology, Inc.) and its corresponding secondary antibody, HRP-conjugated polyclonal goat anti-mouse IgG (1:2,000; Santa Cruz Biotechnology, Inc.) served as a control. Protein-antibody complexes were detected with an enhanced chemiluminescence system (Keygen Biotech Co., Ltd.). Protein band sizes were estimated using AlphaView 2.2.14407 software (ProteinSimple, Santa Clara, CA, USA). The density was correlated to the protein expression and normalized to GAPDH.