Toll-like receptor 4-mediated inflammation triggered by extracellular IFI16 is enhanced by lipopolysaccharide binding
Fig 7
IFI16 binds to TLR4 in vitro and in vivo.
(A) 786-O cells were stimulated for 1 h in the presence or absence of the indicated concentrations of IFI16, LPS from E. coli O111:B4 (LPS-EB), or IFI16/LPS-EB complex. Total cell extracts, untreated or DNase I-treated, were subjected to immunoprecipitation using a TLR4 monoclonal antibody. Immunoprecipitates and whole-cell lysates were analyzed by immunoblotting with anti-IFI16, anti-TLR4 or anti-MD2 antibodies. β-actin protein expression was used for protein loading control. Data are representative of three independent experiments with similar results. (B) Surface plasmon resonance (SPR) analyses of IFI16 binding to immobilized TLR4. After immobilization of TLR4 on the CM5 sensor chip surface, increasing concentration of IFI16 (31.25–1,000 nM) diluted in running buffer were injected over immobilized TLR4. IFI16 binds to TLR4 with an equilibrium dissociation constant (KD) of 0.13 μM. Data are representative of three independent experiments.