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. 2011 May 4;286(26):23334–23344. doi: 10.1074/jbc.M110.205419

FIGURE 1.

FIGURE 1.

Replacement of the ectodomain of TLR4 with LPS-binding proteins leads to constitutive activation. A, schematic representation of the MD-2-TM-TLR4, MD-2-(4CYS)-TM-TLR4, CD14-TM-TLR4, and CD14-(4CYS)-TM-TLR4 chimeric constructs. The conserved cysteine residues of the TLR4 juxtamembrane region are represented as small black circles juxtaposed to the membrane. B, constitutive activation of the NF-κB promoter by the MD-2- and CD14-based TLR4 chimeric constructs. HEK293 cells were transiently transfected with reporter plasmids as well as increasing amounts (1 ng, 5 ng, and 10 ng) of plasmid DNA encoding the MD-2-TM-TLR4, MD-2-(4CYS)-TM-TLR4, CD14-TM-TLR4, or CD14-(4CYS)-TM-TLR4 constructs or with MD-2- and TLR4-encoding plasmids (1 ng each). After 18 h of stimulation with LPS, luciferase activity (RLA) was measured in the cell lysates. C, expression levels of chimeric constructs in HEK293T cells. HEK293T cells were transiently transfected with 2 μg of MD-2-TM-TLR4, MD-2-(4CYS)-TM-TLR4, CD14-TM-TLR4, CD14-(4CYS)-TM-TLR4, or TLR4 plasmid DNA. 48 h post-transfection, cell lysates were prepared, and 30 μg of total proteins were loaded onto an SDS-PAGE gel and blotted using anti-FLAG antibody. Molecular weight marker sizes are given in kDa. D, MD-2- and CD14- based TLR4 chimeric constructs are expressed at the cell surface. HEK293T cells were transiently transfected with 2 μg of MD-2-TM-TLR4, MD-2-(4CYS)-TM-TLR4, CD14-TM-TLR4, or CD14-(4CYS)-TM-TLR4 plasmid DNA. Cell surface expression levels of chimeric constructs were determined by flow cytometric analysis 48 h post-transfection. Histograms of mock-transfected cells (filled areas) or cells transfected with the appropriate DNA (solid lines) are shown. E, MD-2- and CD14-based TLR4 chimeric constructs bind LPS. HEK293T cells were transiently transfected with 2 μg of MD-2-TM-TLR4-, MD-2-(4CYS)-TM-TLR4-, CD14-TM-TLR4-, or CD14-(4CYS)-TM-TLR4-encoding plasmids or with TLR4 and MD-2 plasmid DNA (1 μg each). 48 h post-transfection, binding of FITC-LPS by the receptors was assessed by flow cytometric analysis. Histograms of mock-transfected cells treated with LPS (filled areas) or transfected cells treated with LPS (solid lines) are shown.