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. 2005 Mar 21;201(6):1007–1018. doi: 10.1084/jem.20041442

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Copyright © 2005, The Rockefeller University Press

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Figure 3. — A46R inhibits TLR4 signaling and interacts with TLR4, Mal, and TRAM. (a) HEK 293 cells were transfected with 50 ng CD4-TLR4, 25–100 ng A46R, or pcDNA3.1 (EV) and the NF-κB (left), p38 (middle), or ERK (right) reporter plasmids as indicated. Cells were harvested 24 h after transfection, and luciferase reporter gene activity was measured. (b–d) HEK 293T cells were transfected with A46R and Flag-TLR4 (b), Flag-Mal (c), or Flag-TRAM (d) as indicated. After 24 h, lysates were subject to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. (e) HEK 293T cells were transfected with 8 μg of Flag-TLR4 (top), Flag-Mal (middle), or Flag-TRAM (bottom). After 24 h, lysates were incubated with GST-A46R (lane 3) or GST alone (lane 2), and together with whole cell lysate (lane 1), were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. (f) HEK-TLR4 cells were transfected with the indicated amounts (ng) of A46R 24 h before stimulation with 1 μg/ml LPS, and 24 h after stimulation, supernatants were harvested and assayed for IL-8 by ELISA.