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. Author manuscript; available in PMC: 2017 Aug 15.
Published in final edited form as: J Immunol. 2016 Jul 15;197(4):1343–1352. doi: 10.4049/jimmunol.1502357

Figure 2. TLR9471-1032 does not respond to CpG DNA.

Figure 2

A) A TLR9-deficient macrophage cell line was retrovirally transduced with empty vector, full-length TLR9-HA, or TLR9471-1032 tagged with either HA or myc at the C-terminus. Transduced cells were replated at 2X10^5 cells per well in 24 well plates than treated with media, 3 μM CpG DNA, or 100 ng/ml LPS for six hours in quadruplicate. Supernatants were assayed for TNF-α by ELISA. This experiment was performed 3 times. B) A TLR9-deficient macrophage cell line was retrovirally transduced with empty vector, TLR9, or TLR9471-1032 for 24 hours. Cells were stimulated with 3 μM CpG DNA, or 100 ng/ml LPS for six hours and 10 μg/ml 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. This experiment was performed 3 times. C) Primary mouse bone marrow-derived macrophages (BMM) and dendritic cells (BM-DC) were similarly reconstituted, stimulated and stained as in (B). This experiment was performed 2 times.