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. Author manuscript; available in PMC: 2017 Mar 14.
Published in final edited form as: J Immunol. 2016 Jan 29;196(5):2309–2318. doi: 10.4049/jimmunol.1502074

FIGURE 4. Cell activation by LPS, mediated by MD-2 variants via soluble proteins or cell co-culture.

FIGURE 4

A, and B, HEK293 cells were transiently transfected with plasmids encoding MD-2. After 24 h, harvested medium containing sMD-2 was incubated with lipid A (50, 200 or 500 ng/ml) and added to HEK293 cells that had been transfected with a plasmid encoding h or mTLR4 together with NF-κB-dependent luciferase reporter plasmid. C, D, E, and F, Co-culture of HEK293 cells transfected with plasmids encoding wt or V135A hMD-2 and cells transfected with TLR4 and NF-κB-dependent luciferase reporter plasmids. Co-cultured cells were stimulated with increasing concentration of S-LPS or lipid A for 16 h. G, Inhibition of TLR4 signaling with soluble V135A hMD-2. Culture medium of HEK293 cells transfected with plasmids encoding wt hMD-2 together with V135A hMD-2, in ratios from 1:0 to 1:10, was incubated with lipid A (10, 500 or 1000 ng/ml) and then added to HEK293 cells that had been transfected with a plasmid encoding hTLR4 together with NF-κB-dependent luciferase reporter plasmid. All the results shown are means ± SEM RLA from three independent experiments.