FIGURE 1.

Insect-derived conditioned medium containing secreted MD-2-His contains more active MD-2 and more MD-2 in a monomeric state than HEK-derived MD-2-His. A, functional MD-2 was assayed by measuring activation of HEK/TLR4 cells induced by incubating cells with [¹⁴C]LOS·sCD14 (2 nm) and increasing amounts of conditioned medium (insect, ○; HEK-293, •) for 24 h. Cell activation was monitored by extracellular accumulation of IL-8, which was measured by ELISA. The results shown represent the means ± S.E. of three experiments, each in triplicate. B, composition of secreted MD-2 determined by SDS-PAGE ± dithiothreitol (DTT)/immunoblots (25 μl/lane) harvested at 48 h from HEK293T cells transfected with expression vector (pEF-BOS) encoding MD-2-FLAG-His₆ and cultured in serum-free medium with 0.4% HSA (lanes 1 and 2) or from insect cells infected with baculovirus containing MD-2-His (lanes 3 and 4). MD-2 was detected using anti-(His)₄ antibody. Lane 1 represents molecular weight markers (Perfect Protein; Novagen). The blot shown is representative of more than three experiments.