FIG 5.

Recognition of VpOmpU by TLR1/2 in THP-1 cells and by TLR1/2 and TLR2/6 in RAW 264.7 cells. (A to C) In RAW 264.7 cells, VpOmpU associates with TLR2, TLR1, and TLR6 as evidenced in coimmunoprecipitation (co-IP) and reverse co-IP studies using anti-TLR2, anti-TLR1, and anti-TLR6 antibodies. (A) TLR1 and TLR6 were coimmunoprecipitated with TLR2 in VpOmpU-treated RAW 264.7 cells as observed by immunoblotting (IB) and densitometric analysis of the bands. (B) TLR2 was coimmunoprecipitated with TLR1 in VpOmpU-treated RAW 264.7 cells as observed by immunoblotting and densitometric analysis of the bands. (C) TLR2 was coimmunoprecipitated with TLR6 in VpOmpU-treated RAW 264.7 cells as observed by immunoblotting blotting and densitometric analysis of the bands. (D to F) VpOmpU associates with TLR1 and TLR2 in THP-1 cells as observed by co-IP and reverse co-IP using anti-TLR2, anti-TLR1, and anti-TLR6 antibodies. (D) TLR1 was coimmunoprecipitated with TLR2 in VpOmpU-treated THP-1 cells as observed by immunoblotting and densitometric analysis of the bands. (E) TLR2 was coimmunoprecipitated with TLR1 in VpOmpU-treated THP-1 cells as observed by immunoblotting and densitometric analysis of the bands. (F) TLR2 did not coimmunoprecipitate with TLR6 in VpOmpU-treated THP-1 cells as observed by immunoblotting and densitometric analysis. For panels A to F, cell lysates were prepared after 15 min of VpOmpU treatment and immunoprecipitated with anti-TLR1, anti-TLR2, or anti-TLR6 antibody, and the IP lysates were further analyzed for TLRs coimmunoprecipitated together to identify the receptor complex. For the densitometric analysis, the band intensities of TLR1, TLR2, TLR6, or VpOmpU in the VpOmpU-treated samples above the respective band intensities in the buffer-treated samples were estimated. Results are expressed as mean ± SEM from three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, P > 0.05 [versus band intensities in the buffer-treated cells]). (G) Decrease in TNF-α production with anti-TLR1, but not anti-TLR6, neutralizing antibody in VpOmpU-treated THP-1 cells compared to isotype-pretreated VpOmpU-treated cells and cells treated only with VpOmpU. Results are expressed as mean ± SEM from three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, P > 0.05 [versus cells treated with VpOmpU only]). (H) No decrease in TNF-α production in THP-1 cells with knockdown of TLR6 by siRNA, compared to the nontargeted siRNA-transfected control, in response to VpOmpU. (I) Decrease in TNF-α production with siRNA-mediated knockdown of TLR6, but not TLR1, compared to the nontargeted siRNA-transfected control in RAW 264.7 cells in response to VpOmpU. For panels H and I, results are expressed as mean ± SEM from three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, P > 0.05 [versus VpOmpU-treated cells transfected with nontargeted siRNA]).