FIGURE 3.

LPS or lauric acid induces, but DHA inhibits the homodimerization of TLR4. A, Ba/F3 whole cell lysates were immunoprecipitated with an anti-GFP antibody and then immunoblotted with an anti-FLAG antibody. The membrane was stripped and reprobed with anti-GFP. B, dose-dependent response of Ba/F3 cells toward pretreatment with DHA. Cells were stimulated with LPS or C12, and cell lysates were processed as described in Fig. 2A. For the analysis of NF-κB promoter activity, Ba/F3-TLR4 cells transfected with NF-κB- luciferase construct (C) or RAW264.7 cells transfected with NF-κB- luciferase construct (D) were used. Cells were treated with LPS or lauric acid for 8 h in the presence or absence of DHA. Cells were lysed to determine the luciferase activity. The results were expressed as relative luciferase activity (RLA) against the value of the vehicle treatment. Values are means ± S.E. of the mean of at least three independent experiments. a and b were significantly different from the values for the control group without DHA treatment (p < 0.05). For the analysis of mRNA levels of indicated genes, Ba/F3-TLR4 cells (E) or RAW264.7 cells (F) were treated with LPS or lauric acid in the presence or absence of DHA for 6 h. RNAs were prepared, and reverse transcription-PCR was performed as described under “Experimental Procedures.” Hypoxanthine phosphoribosyltransferase (HPRT) was used as a control.