Rabbit anti β actin

Western blot was performed as previously described.²² Total proteins (20 μg) were subjected to SDS‐PAGE and then transferred onto a polyvinyl indene difluoride membrane (PVDF; Millipore, Billerica, MA, USA). Nonspecific binding was blocked with PBST (0.5% Tween 20 in PBS) containing 5% non‐fat milk (Shandong Sparkjade Biotechnology Co., Ltd.) for 1 h at room temperature. The membranes were then incubated overnight at 4°C with individual primary antibodies in PBST containing 1% non‐fat milk (mouse anti‐IL‐6, rabbit anti‐TNF‐α, rabbit anti‐IL‐1β, rabbit anti‐GFAP, 1:1000, Abcam; mouse anti‐GFAP, rabbit anti‐pSTAT3, mouse anti‐STAT3, rabbit anti‐JAK‐1, rabbit anti‐JAK‐2, 1:1000, Cell Signaling Technology, rabbit anti‐β‐actin, Biosynthesis Biotechnology Inc., Beijing, China). Following three washes with PBST, the membranes were then incubated with the secondary antibodies (HRP‐linked anti‐rabbit IgG; HRP‐linked anti‐mouse IgG; 1:2000; Cell Signaling Technology). Then, the proteins were detected by chemiluminescence reagents (Millipore) and observed using a ChemiDoc™ XRS+ Imaging System (Bio‐RAD, Hercules, USA). The protein levels were quantified by densitometry using Image J 1.4.3.67.

Cells were lysed in RIPA buffer containing a protease inhibitor cocktail (Roche, Mannheim, Germany, 11873580001). Protein concentration was determined. Equal amounts of protein were separated by SDS-PAGE and electrophoretically transferred onto a PVDF membrane (Roche, 03010040001). After blocking with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20, the membrane was incubated with specific primary antibodies, followed by incubation with appropriate horseradish peroxidase–conjugated secondary antibodies. Signals were developed using an enhanced chemiluminescence reagent (Millipore, Darmstadt, Germany, WBKLS0500) and captured on an Alpha Innotech Fluor Chem FC2 imaging system (Alpha Innotech, San Leanardo, CA). Antibodies used in this study were: rabbit anti-β-ACTIN (Biosynthesis Biotechnology, Beijing, China, bs0061R, 1:1000), rabbit anti-HMGB1 (Abcam, Hong Kong, China, ab191583, 1:1000), rabbit anti-AMPK/pAMPK (Cell Signaling Technology, Danvers, MA, #2532 / #2531, 1:1000), rabbit anti MAVS (Abcam, ab31334, 1:500) and HRP-conjugated secondary antibodies (Multisciences, Hangzhou, China, GAR007 and GAM007, 1:5000).