Figure 3.

High-mobility group box 1 (HMGB1) B box activates complement cascade. (A) Schematic overview of recombinant HMGB1 proteins used: wild-type (WT) HMGB1, boxes A (aa 1-79) and B (aa 88-162), ΔC-HMGB1 (aa 1-185), and ΔN-HMGB1 (aa 11-215). (B,C) Complement consumption assessment. WT HMGB1 proteins (B) or HMGB1 variants (C) were incubated with the diluted normal human serum (NHS) containing CH₅₀ activity in GVB²⁺ buffer for 30 min at 37°C. After complement consumption by HMGB1, sRBCs were added in the consumed NHS for 30 min at 37°C. Aggregated human IgG and IgG-free BSA (each 30 µg/ml) were used as the positive and negative controls, respectively. Data shown are the mean ± SD of three independent repeats. *P < 0.01 vs. BSA30, **P < 0.001 by two-way ANOVA with Bonferroni correction.