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. 2017 Jun 27;292(32):13415–13427. doi: 10.1074/jbc.M117.784983

Figure 2.

Figure 2.

A peptide derived from the TLR4 TMD inhibited heterodimerization by interacting with its target receptor within the membrane. A and B, the TLR4 and TLR6 TMDs form a heterodimer within a membrane mimicking environment. 0.2 μm TLR6C2 peptide (A) scrTLR4C peptide (B) were labeled with NBD (donor) and added to 100 μm PC:Chol LUVs in PBS buffer. Changes in the intensity of the emission signal were monitored between 500 and 600 nm upon the addition of successive amounts of rhodamine labeled TLR4C peptide (acceptor). Acceptor peptide was added at ratios of 1:32 to 1:4. For each experiment the spectra was normalized to the value of the donor alone. Curves were calculated based on the average of three independent experiments. C and D, the TLR6C2 peptide interacts with the LUVs. C, 0.2 μm TLR6C2 peptide was labeled with NBD, and PC:cholesterol LUVs were added at different peptide lipid ratios: 1:2000, 1:1500, 1:1000, 1;750, 1:500, 1;250, 1:100. Changes in the intensity of the emission signal were monitored between 500 and 600 nm. D, the Kd of the TLR6C2 peptide was calculated. Curves were calculated based on the average of three independent experiments. E, the TLR4C peptide physically interacted with its corresponding receptor, TLR6. BV2 microglia cells were incubated with 20 μm concentrations of rhodamine-labeled peptide for 2 h at 37 °C. Then the cells were lysed using radio RIPA, and the soluble fraction was used for immunoprecipitation with antibodies against TLR6 or TLR2. As a negative control we used the scrTLR2C peptide. Protein samples were run on SDS-PAGE, and the presence of the peptide was detected with a fluorescence scanner (excitation at 532 nm and emission at 585 nm). Nonspecific binding of the peptides to G protein beads was subtracted. Subsequently, the gel was transferred to a membrane and subjected to Western blotting (W.B.) for TLR6 and TLR2 in the appropriate samples. Equal loading was measured by detecting of anti-tubulin in the cell lysate. Results are presented as three independent experiments. A.U., absorbance units.