Figure 6.

TTN blocked lipopolysaccharide (LPS)-induced TLR4 dimerization involving the MyD88 pathway. (a) HEK293T cells were co-transfected with TLR4-HA and TLR4-Flag for 24 h. Cells were pretreated with TTN (4 μg/ml) for 1 h before LPS (1 μg/ml) stimulation for another 24 h. The extent of TLR4 dimerization was determined by co-immunoprecipitation assay. Collected proteins were immunoprecipitated with Anti-HA Magnetic Beads. Immunocomplexes were determined by western blotting analysis with anti-HA and anti-Flag antibodies (n=5). (b) Quantification of TLR4-HA and TLR4-Flag in HEK293T cells. Quantification of TLR4-HA and TLR4-Flag was detected by densitometric analysis, and TLR4-Flag was compared with TLR4-HA. (c) RAW264.7 cells were pretreated with TTN (4 μg/ml) for 1 h before LPS (1 μg/ml) stimulation for another 2 h. Collected proteins were immunoprecipitated with MyD88 using magnetic beads. Immunocomplexes were determined by western blotting analysis with anti-MyD88 and anti-TLR4 antibodies (n=5). (d) Quantification of MyD88 and TLR4 was detected by densitometric analysis, and TLR4 was compared with MyD88. (e and f) RAW264.7 cells were pretreated with TTN (4 μg/ml) for 1 h before LPS (1 μg/ml) stimulation for another 2 h. The expression of p-TAK1 and TAK1 was examined by western blotting analysis. The values were expressed as means±S.D. **P<0.01 and ***P<0.001 versus LPS alone group