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. 2012 Aug 27;209(9):1519–1528. doi: 10.1084/jem.20120189

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© 2012 Venereau et al

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Figure 2. — The cytokine-stimulating and chemoattractant activities of HMGB1 are mutually exclusive. (A) HMGB1/CXCL12 heterocomplex detected by hybrid ELISA. All-thiol- or disulfide-HMGB1 (7.5 ng) were preincubated with the indicated amount CXCL12 at 37°C for 15 min. The formation of the heterocomplex was detected by hybrid ELISA (an anti-CXCL12 capture antibody and an anti-HMGB1 detection antibody). Results are expressed as absorbance at 450 nm (*, P < 0.05, ANOVA). (B) Human monocyte migration in response to increasing concentrations of CXCL12 in the presence or absence of 300 nM all-thiol- or disulfide-HMGB1 (*, P < 0.01 vs. CXCL12 alone; 2-way ANOVA). (C) Migration of mouse 3T3 fibroblasts toward disulfide-HMGB1 or all-thiol-HMGB1, or disufide-HMGB1 exposed to 5 mM of DTT for 30 min (newly all-thiol-HMGB1; *, P < 0.05 vs. disulfide-HMGB1, ANOVA). (D) Migration of 3T3 fibroblasts toward all-thiol-HMGB1 in the presence of increasing concentrations of disulfide-HMGB1, and expression of TNF (as fold increase compared with unstimulated macrophages) in human macrophages stimulated for 4 h with disulfide-HMGB1 in the presence of increasing concentrations of all-thiol-HMGB1. The effects of the competing form of HMGB1 are not statistically significant (ANOVA). (E and F) Migration of mouse 3T3 fibroblasts toward WT all-thiol-HMGB1 previously exposed to increasing concentrations of H₂O₂ for 1 h (E; *, P < 0.05 vs. all-thiol HMGB1 not treated with H₂O₂, ANOVA), and toward WT all-thiol-HMGB1 or the E106 mutant purified in the presence of DTT (F; *, P < 0.05 vs. untreated control). (G) Human macrophages were stimulated for 4 h with WT disulfide-HMGB1 or the E106 mutant (0.4 µM) prepared in the absence of DTT. Expression of TNF, MIP-2, and IL-8 was measured by real-time PCR and expressed as fold increase compared with unstimulated macrophages (*, P < 0.05 vs. disulfide-HMGB1; Student’s t test). In all panels, data are representative of at least three independent experiments and bars represent the mean ± SD of triplicate samples (when not visible, they fall within symbols).