Standard cell lysis buffer

Cytosolic proteins were isolated using a standard cell lysis buffer (Cell Signaling Technology Inc., Danvers, MA, USA), and supplemented with a protease inhibitor cocktail (Roche Biochemicals, Basel, Switzerland). Protein samples were separated on a gel (12%) using sodium dodecyl sulfate polyacrylamide gel electrophoresis; these were transferred to a polyvinylidene difluoride hydrophobic membrane (Millipore, Bedford, MA, USA). Nonspecific binding sites on the membrane were blocked overnight with 5% skimmed milk (Solarbio, Beijing, China) at 4°C; the membrane was then inoculated with a rabbit polyclonal antibody against TLR-4 (Abcam, Cambridge, UK) or GAPDH (Sinobio, Beijing, China) at room temperature for 2 h; subsequently, the membrane was stained with a peroxidaseconjugated secondary antibody against rabbit IgG (Pierce, Rockford, IL, USA) at room temperature for 1 h. Finally, specific binding was visualized by chemiluminescence by using the enhanced chemiluminescence western blotting detection reagent (Amersham, Uppsala, Sweden). TLR-4 expression (gray color) was determined relative to that of GAPDH.