Western Blotting of Brain Proteins

Western blotting of proteins of freshly collected brains was performed as described [23 (link),24 (link)]. The primary antibodies were polyclonal rabbit anti-β-actin (Proteintech, 20536-1-AP, 1: 1000, Rosemont, IL, USA), polyclonal rabbit anti-TLR4 (Abcam, ab13556, 1: 500, Cambridge, UK), polyclonal rabbit anti-Myd88 (Abcam, ab2064, 1: 500), polyclonal rabbit anti-HMGB1 (Abcam, ab18256, 1: 200) and polyclonal rabbit anti-NF-κB (Cell Signaling Technology, CST, #8242, 1: 500, Danvers, MA, USA), all diluted with phosphate buffered saline (PBS). The membranes were washed with PBS and incubated with goat anti-rabbit IgG secondary antibody (ZSJQ, ZB-2308, Beijing, China). Relative protein expression was assessed densitometrically with a BD imaging system (Becton Dickinson, Franklin Lakes, NJ, USA).