Anti hmgb1 antibody

Cytosolic and nuclear proteins from the ischemic cortex were prepared with the Nuclear/Cytosol Fractionation Kit (BioVision, Mountain View, CA, USA) for western blotting analysis. As previously described in detail, the protein samples were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane (Millipore Corporation, Billerica, MA, USA). The membrane was incubated with the following antibodies: anti-HMGB1 antibody (diluted 1:1,000, Cell Signaling Technology), anti-TLR4 antibody (diluted 1:200, Abcam), anti-NFκB p65 antibody (diluted 1:500; Abcam), anti-IκB antibody (diluted 1:500, Abcam), anti-phospho-IκB antibody (diluted 1:500; Abcam), and phospho-NFκB p65 antibody (diluted 1:500; Abcam). To confirm equal loading, we used an anti-GAPDH antibody (1:500 dilution, Santa Cruz Biotechnology) and an anti-lamin A antibody (diluted 1:1,000, Abcam). The density of each band was quantified using ImageJ.

This study was approved by the Laboratory Animal Center of the Academy of the China Pharmaceutical University. 50 male healthy Sprague-Dawley rats weighing from 200 to 220 g were purchased from the Animal Experimental Animal Center of Jilin University. Animal experiment was carried out in accordance with the Guidelines for Animal Experiment of Jilin University. The animals were maintained in a temperature controlled room at 20.1–23.1°C and 40–50% humidity, a 12 h light/dark cycle, and free access to water. Berberine with purity of over 98% was purchased from National Institutes for Food and Drug Control (Beijing, China). Isoproterenol was purchased from Shanghai Hefeng Pharmaceutical Co. Ltd. (Shanghai, China). Sodium pentobarbital was purchased from Merck (Germany). Propranolol was purchased from Xi'an Li Jun Pharmaceutical Co. ltd. (Xi'an, China). CK-MB, LDH, T-SOD, MDA, TNF-α, and IL-6 test kits were all purchased from Nanjing Jian Cheng Biological Engineering Research Institute (Nanjing City, China): anti-HMGB1 antibody (Cell Signaling Technology, #3935), anti-TLR4 antibody (Cell Signaling Technology, #2246), anti-TNF-α antibody (Cell Signaling Technology, #6945), anti-Bax antibody (Cell Signaling Technology, #2272), anti-Bcl-2 antibody (Cell Signaling Technology, #2876), and anti-GAPDH antibody (Cell Signaling Technology, #5174).