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. Author manuscript; available in PMC: 2014 Oct 1.
Published in final edited form as: Innate Immun. 2013 Feb 25;19(5):545–560. doi: 10.1177/1753425913475688

Figure 3. Comparison of [3H]LOS·MD-2 (A, C) and LOS·MD-2[125I] (B, D) dose-dependent binding to and activation of membrane-bound TLR4 in transiently transfected HEK293T cells.

Figure 3

Binding of increasing concentrations of [3H]LOS·MD-2 (A) and LOS·MD-2[125I] (B) to HEK293T cells (2 million cells/dose) transiently transfected (Lipofectamine2000) with TLR4 or control vector during 30 min incubation at 37°C. Inserts in A and B show dose-dependent [3H]LOS·MD-2 (A) and LOS·MD-2[125I] (B) bound after incubation with TLR4 transfected cells (total) and mock transfected cells (non-specific). Specific binding shown in A and B as a saturation curve was calculated as the difference between total and non-specific cpm bound. Scatchard analyses of these data by GraphPad Prism were used to determine Kd and Bmax. Binding to transiently transfected TLR4 cells indicated Kd = 367 ± 287 pM for [3H]LOS·MD-2 and Kd = 694 ± 101 pM for LOS·MD-2[125I]. Binding experiments were performed as described in Material and Methods. Data shown represent a composite of more than three experiments, each dose done in duplicate, using aliquots of the same cell preparation for both [3H]LOS·MD-2 (A) and LOS·MD-2[125I] (B) for binding. Bmax ranged from 26–51 pM, corresponding to an average of 8,000–15,000 cell surface TLR4/cell. Dose-dependent cell activation by [3H]LOS·MD-2 (C) and LOS·MD-2[125I] (D) on aliquots of the transiently transfected TLR4 or mock transfected cells used in binding experiments in (A) and (B) was measured as extracellular accumulation of secreted IL-8 by ELISA as described in Materials and Methods. Data shown are from one experiment with duplicate samples for each dose and are representative of more than three similar experiments.