Western Blotting Analysis of iNOS and COX-2

The Western blotting analysis was performed as previously described [31 (link)]. Briefly, the samples with cell lysates were boiled for 10 min at 100 °C. The concentrations of proteins in the cell lysates were quantified using the bicinchoninic acid method. Equal amounts of protein were subjected to 8%–10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA) activated with 100% methanol. The membranes were blocked using 5% BSA in a mixture of Tris-buffered saline and Tween 20 (1×), and subsequently probed with the following antibodies: anti-iNOS, COX-2, and GAPDH (Cell Signaling Technology, Beverly, MA, USA). The blots were visualized using an enhanced chemiluminescence detection kit (Intron Biotechnology, Sungnam, Korea) and an ImageQuant LAS-4000 Imager (Fujifilm Corp., Tokyo, Japan).