Immunoprecipitation assay buffer

Brain tissues were lysed and homogenized in immunoprecipitation assay buffer (Beyotime, Cat# P0013D) with protease inhibitor (BB-3301, Bestbio, Nanjing, China). Sections were then analyzed using kits for mouse IL-18 (Andy Gene, Beijing, China, Cat# AD1905Mo), mouse IL-1β (Andy Gene, Cat# AD3364Mo), mouse IL-6 (Jiangsu Meibiao Biotechnology Co., Ltd., Nanjing, China, Cat# MB-2899A) and mouse IL-4 (Jiangsu Meibiao Biotechnology Co., Ltd., Cat# MB-3400A) following the manufacturer’s instruction.

After completion of cell processing, in order to obtain whole-cell lysates, the cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed on ice with immunoprecipitation assay buffer (Beyotime Biotechnology) containing a protease inhibitor cocktail. Then, these cellular lysates were centrifuged at 13,000 rpm for 10 min at 4°C. Whole-cell lysates samples were boiled at 100°C for 10 min with loading buffer (5% SDS, 10% glycerol, 60 mM Tris pH 6.8, 5% β-mercaptoethanol, and 0.01% bromophenol blue). Protein samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in 5% non-fat milk-TBST for 3 h at room temperature and incubated with primary antibodies overnight followed at 4°C. Bound antibodies were visualized by HRP-conjugated secondary antibodies for 1.5 h at room temperature and enhanced chemiluminescence (ECL) system (Advansta) with a Kodak imager. All data are representative of three independent experiments with triplicate samples. The quantitative analysis of the relative intensities of proteins (normalized to β-actin) was performed with Quantity One Software (Bio-Rad) and GraphPad Prism 5.