Figure EV1. Binding of DFL to HMGB1 assessed by NMR and chemotaxis experiments on R23A and R109A HMGB1 mutants.

• A
  Saturation transfer difference (STD) results obtained for 1 mM DFL alone (black line) and with 0.05 mM HMGB1 (red line) in 20 mM phosphate buffer, 150 mM NaCl, 1 mM DTT pH 7.3 (on resonance: 0 ppm; saturation time: 3 s). STD signals with different intensities are observed for all protons in the presence of HMGB1, indicating that DFL directly binds to HMGB1. The numbered peaks correspond to proton resonance assignments.
• B
  WaterLOGSY spectra obtained for 1 mM DFL alone (black line) and in complex with 0.05 mM HMGB1 (red line). All DFL protons display signals inversion, indicating binding to HMGB1.
• C
  Superimposition of the ¹H‐¹⁵N HSQC spectra of HMGB1 (˜0.1 mM, pH 7.3, phosphate buffer, 1 mM DTT) without (black) and with 10‐fold excess of DFL (red). Inset: selected region of the superimposition of ¹H‐¹⁵N HSQC spectra of HMGB1 during the titration with DFL (0.5, 1, 2, 3, 5, and 10 equivalents) showing the displacement of the peak associated to R23 along the titration. On the right: weighted average of R23 and V124 amide ¹H and ¹⁵N chemical shift changes in the presence of increasing concentration of DFL. Nonlinear curve fitting yields K _d = 2.8 ± 1.4 mM.
• D, E
  The R23A (D) and R109A (E) HMGB1 mutants have chemotactic activity but are not inhibited by DFL. Data points with average ± standard deviation (avg ± SD; n = 3, each point represents a biological replicate) in one representative experiment (of two experiments performed in different days). Statistics: one‐way ANOVA (P < 0.0001), followed by Dunnett's post‐tests. *P < 0.05 relative to no DFL addition. The difference in migration toward R109A HMGB1 in the presence of increasing concentrations of DFL is statistically non‐significant (one‐way ANOVA plus post‐tests).

Source data are available online for this figure.