Fig. 3

CitH3 inductions in blood PMNs by HMGB1 treatment. a-b HMGB1 levels in blood and CSF were determined after 6 h to 4 d of MCAO by immunoblotting. c-d CitH3 levels were examined after treating neutrophils isolated from peripheral blood with all-thiol HMGB1 or disulfide HMGB1 (0.5 or 1 μg/ml) for 1, 2, 3, and 4 h or for 1, 2, 4, and 6 h, respectively, by immunoblotting. e Blood neutrophils were treated with all-thiol HMGB1 or disulfide HMGB1 (0.5 or 1 μg/ml) for 2 or 4 h and double fluorescent staining was conducted using anti-CitH3 antibody plus DAPI. f-g CitH3 levels were examined by immunoblotting after treating blood neutrophils with 0.5 μg/ml of all-thiol HMGB1 (f) or disulfide HMGB1 (g) for 4 h with or without pre-treating circulating neutrophils with AMD3100 (10 μM), TLR-IN-C34 (10 μM), or PAD inhibitor (10 μM) for 30 min. Scale bars in E represent 100 μm