Figure 4.

The recruitment of TLR4 to lipid rafts was inhibited by platycodin D (PLD). Cells were pretreated with PLD or MβCD, followed by treatment with LPS. The cells were lysed and subjected to discontinuous sucrose density gradient centrifugation as described in Section “Materials and Methods.” The fractions were analyzed by using CTxB conjugated to horseradish peroxidase (GM1) or anti-TLR4 primary antibody by western blotting. Fractions 3–4 correspond to lipid rafts. Representative blots of three separate experiments are shown. TLR4 content of macrophage lipid rafts was calculated as a percentage of total membrane TLR4 (lipid rafts + nonrafts). The values presented are the means ± SD of three independent experiments and differences between mean values were assessed by one-way ANOVA with Tukey multiple comparison test (^#p < 0.05 vs. control group; *p < 0.05, **p < 0.01 vs. LPS group).