Figure 4.

Single mutation at Cys747 in TLR4 affects LPS-induced NF-κB activation. (A) Binding of [³H]-TAK-242 to TLR4 mutants with single mutation at Cys747 in TLR4. COS-7 cells were transiently transfected with expression vectors coding FLAG-TLR4 and FLAG-TLR4 with single amino acid mutations in TLR4. After 2 days of transfection, the cells were incubated with 100 nM of [³H]-TAK-242 for 6 h. Cell lysates were immunoprecipitated with anti-FLAG M2 Ab, and the immunoprecipitates were subjected to SDS-PAGE and western blotting. Radioactive imaging of the immunoprecipitates was analysed by autoradiography. Data shown are representative of two independent experiments. (B) Effect of TAK-242 on LPS-induced NF-κB activation in TLR4 mutants with single mutation at Cys747 in TLR4. HEK293 cells were transiently transfected with expression vectors coding TLR4 and TLR4 with single amino acid mutations along with pNifty and phRL-TK. Expression vectors coding MD-2 and CD14 were also co-transfected. On the day after transfection, cells were incubated with 100 ng·mL⁻¹ LPS and various concentrations of TAK-242 for 6 h. Luciferase activity was determined using a luciferase assay kit. Data represent the mean ± SEM in triplicate wells. Data shown are representative of three independent experiments. HEK, human embryonic kidney; LPS, lipopolysaccharide; NF-κB, nuclear factor-κB; TLR, Toll-like receptor.