FIGURE 7.

The R753Q polymorphism impairs the ability of TLR2 to hetero-dimerize with TLR6 upon Pam2Cys stimulation. A, 293/YFP-TLR2 stable cell lines expressing WT or R753Q YFP-TLR2 variants were co-transfected with pcDNA3-CFP-TLR6. After recovery for 20 h, cells were treated for the indicated time points with medium or 100 ng/ml Pam2Cys. Whole cell lysates were prepared, and TLR2 species were immunoprecipitated (IP) with anti-TLR2 Ab (TL2.1) and analyzed by immunoblotting (IB) with anti-TLR6 Ab to detect TLR2-associated TLR6. Total levels of transfected YFP-TLR2 and CFP-TLR6 proteins were assessed by Western blot analyses of whole cell lysates with anti-TLR2 and anti-TLR6 Abs, respectively. Immunoblotting of cell lysates with anti-tubulin Ab was used to control for protein loading. The results of a representative (n = 3) experiment are shown. B, quantification of the results shown in A. TLR2-TLR6 arbitrary units were calculated by measuring densitometric values for TLR2-TLR6, TLR2, TLR6, and tubulin at each time point, subtracting background values, normalizing for tubulin, and then dividing TLR2-TLR6 values by those calculated for TLR2 and by TLR6.