Fig. 1

The C-terminal acidic tail domain inhibits binding of HMGB1 to TLR2. a) Binding of HMGB1 to TLR2 was investigated by ELISA. Plates were coated with different batches of HMGB1 and incubated with increasing concentrations of TLR2-Fc. No interaction between commercial HMGB1 and TLR2-Fc was detected. In contrast, in-house produced HMGB1 bound to TLR2-Fc in a dose-dependent manner. Ds-HMGB1 = disulfide HMGB1, Fr-HMGB1 = fully reduced HMGB1. b) SDS-page gel electrophoresis analysis of the in-house and commercial proteins confirmed that the commercial preparation only contained full length HMGB1 whilst the in-house preparation was a mixture of full length and C-terminus truncated protein. c) Schematic structure and SDS-page gel analysis of full length and C-terminal truncated HMGB1 proteins (Δ18 and Δ30) d) Δ30 and Δ18 with a full or partially truncated C-terminus bind to TLR2-Fc as detected by ELISA e) Increasing concentrations of Δ30 results in increasing binding to TLR2-Fc. f-h) Control experiments to confirm that the interaction is specific to TLR2 (f), is not due to differences in coating of the recombinant proteins to the ELISA plates (g) and can be inhibited using enzymatic digestion of the Δ30 protein (h). Representative data shown from 3 to 5 experiments. In e, f and g BSA is represented by an opened triangle