FIGURE 3.
Signaling induced by hexaacylated lipid A through TLR4 requires higher expression levels of the co-receptor, MD-2. Two × 105 cells/well HEK293T cells were co-transfected with untagged wild-type TLR4 (300 ng/well), huCD14 (30 ng/well) and the indicated amounts of (A). huMD-2 (3 ng/well), (B). huMD-2 (300 ng/well) along with the reporter constructs. After overnight recovery, transfected cells were treated with the indicated concentrations of E. coli K235 LPS or S. flexneri 2a LPS or hexaacylataed or pentaacylated lipid A for 5 hr. Luciferase and β-galactosidase activities were measured in cell lysates as described in the Methods. A representative experiment is shown (n = 5). (C). Murine MD-2 rescues pentaacylated signaling by human TLR4. Experiments were set up as described in (A) and (B) using either human or murine MD-2 (100 ng/well) in conjunction with human TLR4 and CD14 constructs. A representative experiment is shown (n = 3).