Polyvinylidene fluoride membranes

RAW 264.7 macrophages were treated as same as RT-PCR. The cells were gently washed twice with ice-cold PBS, and lysed in Pierce^TM RIPA Buffer (Thermo Fisher, San Diego, CA, USA). Proteins were collected by centrifugation at 4 °C, 12,000× g for 10 min. Total proteins content was determined using a Pierce^TM BCA Protein Assay Kit (Thermo Fisher, San Diego, CA, USA). Subsequently, proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene fluoride membranes (Biosharp, Beijing, China). The membranes were blocked with 5% nonfat milk for 2 h at room temperature. The blocked membranes were then incubated with the primary antibodies recognizing TLR4 (Proteintech, Rosemont, IL, USA), MyD88 (Cell Signaling Technology, Danvers, MA, USA), p65, p-p65, IκBα, p-IκBα, iNOS and COX-2 (Abcam, Cambridge, UK), The reaction was allowed to proceed, at 4 °C for overnight, after which the membranes were incubated with secondary antibodies for 1.5 h at room temperature. The immunoreactive proteins on the blots were detected using an enhanced chemiluminescence detection system (Tanon, Shanghai, China). β-actin (Cell Signaling Technology, Danvers, MA, USA) was used as the internal control, and band intensities were quantified using Image-J.