Figure 3. The C terminus is required for binding of HMGB1 to RAGE but not to the α_Vβ₃ integrin.

HMGB1-Flag or ΔC-HMGB1-Flag (50 ng) was incubated with 100 ng soluble α_Vβ₃ (A) or soluble RAGE (sRAGE)-Fc chimeric protein (B). α_Vβ₃- or RAGE-bound HMGB1 or ΔC-HMGB1 was pulled down using agarose beads conjugated with anti-Flag antibody. The precipitated proteins were eluted and resolved by 12% SDS-PAGE, and Western blotting was performed using anti-β₃ antibody (A) or anti-RAGE antibody. (B) Blots were stripped and reprobed with anti-Flag antibody to demonstrate the amount of Flag-tagged HMGB1 or ΔC-HMGB1 that was pulled down by the beads. A second independent experiment provided similar findings. IP, Immunoprecipitation; IB, immunoblotting.