TM, via D1, binds to HMGB1. (A) For IP, rhs-TM (10 nM) and HMGB1 (10 nM) were mixed and incubated, followed by the addition of protein A agarose beads conjugated to anti-HMGB1 IgG (αHMGB1) or nonimmune IgG (IgG). Immunoprecipitates were solubilized, and reduced SDS-PAGE (10%) was followed by immunoblotting with anti-TM antibody (αTM). (B) TM extracellular domains. (C) Competition between TM and sRAGE for binding to HMGB1. First, sRAGE-His (10 nM) was incubated with HMGB1 alone (10 nM; lane 2) or in the presence of rhs-TM (TM, 1 μM; lane 3), P-D1 (1 μM; lane 4), or P-D2+3 (1 μM; lane 5). Then, nickel resin beads were added, precipitates were solubilized, and SDS-PAGE (10%) was followed by IB with αHMGB1 IgG. N, untreated controls. (D) Binding of sRAGE-His (10 nM) to HMGB1 immobilized on plastic plates was studied with untreated controls, TM, P-D1, or P-D2+3. (E) Left panel: Binding of HMGB1-MBP (1 nM) to RAGE-transfected (RAGE) or mock-transfected (Mock) COS-7 cells (10⁴ cells/well) was assessed in the presence of untreated controls, αHMGB1, HMGB1, TM, P-D1, or P-D2+3 (100 nM each). Results shown are representative of 4 replicate wells. The right panel shows RAGE expression in RAGE-transfected versus mock-transfected COS-7 cells by IB (upper) and immunofluorescence on nonpermeabilized fixed cells (lower). *P < 0.05, compared with the control group. ^#P < 0.05, compared with the paired, untreated controls.