Figure 5

The requirement for a disulfide bond between C23–C45 for the cytokinestimulating activity of HMGB1. (A) Effects of mild H₂O₂ exposure (50 µmol/L, 120 min) on the TNF stimulating activity of HMGB1 prepared with DTT. (B) Effects of an A45 mutation of HMGB1 on TNF release from RAW 264.7 cells induced by A45 HMGB1 compared with HMGB1 prepared in the absence of DTT. RAW 264.7 cells were incubated with HMGB1 over a range of concentrations (0 to 10 µg/mL). After 16 h, TNF released into the cell culture supernatant was measured by ELISA. N = 3. *P < 0.05 versus HMGB1. All HMGB1 preparations were characterized by LC-MS/MS prior to cell treatment with respect to cysteine redox status.