Figure 3. Inclusion of the hinge region TLR9441-470 does not restore signaling by TLR9471-1032.
A-B) Primary bone marrow-derived macrophages (BMM) were retrovirally reconstituted with TLR9-HA, TLR9471-1032, or TLR9441-1032 and stimulated with media, 5 μM CpG DNA (A) or 100 ng/ml LPS (B) for six hours. Brefeldin A was added for the final four hours of stimulation. Cells were fixed, permeabilized, and stained for intracellular TNF-α and the HA tag on TLR9. MFI of the HA positive cells is shown. C-D) As in A-B except primary bone marrow derived dendritic cells (BM-DC) were reconstituted by retroviral transduction. E-F) A TLR9 deficient macrophage cell line (TLR9KO line) was reconstituted with empty vector, TLR9, TLR9471-1032, or TLR9441-1032 and stimulated with media, 3 μM CpG DNA (E) or 100 ng/ml LPS (F) for six hours. Supernatants were assayed for TNF-α by ELISA. Each experiment was performed 2 times.