COX-2 and cPLA2 Analysis by Western Blotting

To evaluate the COX-2 and cPLA2 secretion, BV-2 cells were in 6-well plates as aforementioned. When the cells reached 90% confluently, the medium was discarded. We then collected the cells in cold conditions to avoid protein degradation. We lysed the cell by adding 100 μL lysis buffer and protease inhibitor into them at 4 ◦C for 45 min. The tubes were centrifuged at 15,000 rpm for 15 min at 4 ◦C, and the supernatant was

collected and stored for further analysis. The protein content was measured with a Pierce bicinchoninic acid protein assay kit (Pierce Biotechnology). Proteins (20 μg) from each sample were suspended in Laemmli buffer, heated for 5 min at 100 ◦C, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 4~12% gradient. Proteins were electroblotted onto a hydrophobic polyvinylidene difluoride membrane (Pall Corporation, Port Washington, NY, USA). Following transfer, membranes were blocked in 5% bovine serum albumin (BSA) in Tris-buffered saline solution (TBST) with 0.1% Tween-20 for 45 min and then incubated overnight at 4 ◦C with a primary anticyclo-oxygenase (COX)-2 antibody (GTX100656, diluted 1:1000; GeneTex, Alton Pkwy, CA, USA) and a primary anti-cPLA2. After washing three times with 1x TBST, the membranes were probed with a secondary antibody (horseradish peroxidase (HRP)-conjugated antirabbit immunoglobulin G (IgG)) (GeneTex) for one hour and washed three times with TBST. Immunoreactivity was detected using an enhanced chemiluminescence (ECL) kit (GE Healthcare, Chicago, NY, USA) and visualized with the UVP system (Analytic Jena, Upland, CA, USA). Anti-GAPDH from Santa Cruz (Heidelberg, Germany) was used as an internal control. Image J software (1.52 k, Wayne Rasband, NIH) was used to quantify the intensity of the protein bands.
