Fig. 1.

Both the ecto and TIR domains of TLR9 localize intracellularly. A, HEK293T cells were transfected with TLR4-GFP, TLR9-GFP, TLR4-9-GFP, TLR9-4-GFP or TLR9ΔTIR-GFP (TLR9-GFP with the TIR domain deleted). After 24 hr, the cells were stained directly (Surface) or fixed, permeabilized, and stained (Total) with anti-TLR4 or anti-TLR9 mAb (black lines). Cells transfected with GFP and stained identically served as negative controls (filled histograms). Cells were gated for GFP expression and only the GFP⁺ cells are shown. Data are representative of three separate experiments. B, HeLa cells were transfected with the same constructs as in panel A and fixed 24 hours later. GFP fluorescence, differential interference contrast (DIC), and overlays are shown. C, HEK293 cells were transfected with TLR9-4, or TLR9 alone, or with TLR4 plus MD2, or TLR4-9 plus MD2, and stimulated with 100 ng/ml LPS or 5 μg/ml CpG DNA. NF-κB activity was measured 24 hours later. * p<.002, #p <.05. This is one of three replicate experiments.