Interleukin il 1β

Using homogenized buffer (0.25M sucrose, 10mM Tris–HCl, pH 7.4, 0.5mM EDTA, 1mM phenylmethylsulfonyl fluoride, 1mM Na₃VO₄), brain tissue was homogenized and centrifuged twice at 16,300 × g for 15 min at 4°C. A protein assay kit (Pierce Chemical, Rockford, IL, USA) was used to assay samples for protein concentration. Proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline/Tween 20 solution. The blots were incubated with inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2; Millipore Technology Inc., Danvers, MA, USA), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). As an internal control, GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (Santa Cruz Biotechnology, Inc.) was performed. Tris-buffered saline/Tween 20 was used to wash the blots, and then horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology Inc., Beverly, MA, USA) were applied. Blots were then developed using the enhanced chemiluminescence detection kit (GE Healthcare, Buckinghamshire, UK).