Figure 1.

TAK-242 preferentially binds to TLR4, but not to other TLRs, TLR-related adaptor molecules or MD-2. (A) Binding of [³H]-TAK-242 to TLR-related adaptor molecules. HEK293 cells were transiently transfected with expression vector coding FLAG-TRAM, FLAG-TRIF, HA-TIRAP/Mal, HA-MyD88 and FLAG-TLR4. Empty lanes were used for a molecular weight marker. (B) Binding of [³H]-TAK-242 to various TLRs. HEK293 cells were transiently transfected with expression vector coding TLR3-HA, TLR9-HA and FLAG-TLR4. COS-7 cells were transiently transfected with expression vector coding FLAG-TLR2, FLAG-TLR4 and TLR5-HA. Empty lanes were used for a molecular weight marker. (C) Effect of nonradioactive TAK-242 and its enantiomer on the binding of [³H]-TAK-242 to TLR4. COS-7 cells were transiently transfected with expression vector coding FLAG-TLR2 and FLAG-TLR4. (D) Binding of [³H]-TAK-242 to MD-2. COS-7 cells were transiently transfected with FLAG-MD-2, FLAG-TLR2 and FLAG-TLR4. After 2 days of transfection, the cells were incubated with 100 nM of [³H]-TAK-242 for 6 h, then lysed. For competition assay, the cells were incubated with various concentrations of nonradioactive TAK-242 or 10 µM of its enantiomer for 2 h, and with 100 nM [³H]-TAK-242 for 4 h. Cell lysates were immunoprecipitated with anti-FLAG M2 and anti-HA 12CA5 Abs. The immunoprecipitates were subjected to SDS-PAGE and western blotting. Radioactive imaging of the immunoprecipitates was analysed by autoradiography. All data shown are representative of three independent experiments. HEK, human embryonic kidney; Mal, MyD88-adaptor like; TIRAP, TIR domain-containing adaptor protein; TLR, Toll-like receptor; TRAM, TRIF-related adaptor molecule; TRIF, TIR domain-containing adaptor inducing IFN-β.