Figure 4.

Peptides derived from the D helix or subsequent region that includes DE loop, E strand and EE loop of TIR of TLR1 or TLR6 do not block TLR2 co-IP with co-receptors. (A, B) HEK293T cells were transfected with Flag-tagged mouse TLR2 and Cer-tagged mouse TLR1 TIR or TLR6 proteins. Cells were pretreated for 30 min with 20 μM of indicated peptides prior to stimulation with P3C (500 ng ml⁻¹) or P2C (50 ng ml⁻¹) for 20 min. The cell lysates were immunoprecipitated with anti-Flag antibody and the immune complexes assessed using anti-eCFP Ab. CP is a scrambled control peptide. (C) Peptide-protein co-IP assay was performed as described in Fig. 3A. Cell lysates of HEK293T cells that express Flag-tagged mouse TLR2 were immunoprecipitated with anti-Flag Ab. TR9 is a peptide derived from D helix of TIRAP TIR (Couture et al.2012). One representative of three (panel A and B) or two (panel C) individual experiments is shown.