MD-2s fails to mediate LPS-dependent NF-κB activation and IL-8 secretion. MD-2s fails to mediate LPS-dependent NF-κB activation and IL-8 secretion. A, HEK293 cells stably transfected with TLR4 and an NF-κB reporter gene were treated with supernatants containing either MD-2 or MD-2s. Cells were left untreated or stimulated with LPS (250 ng/ml) for 6 h. Mean relative stimulation of luciferase activity ± SD for a representative experiment, each performed in triplicate, is shown. B, Supernatants were collected and measured for IL-8 secretion. C, HEK293 cells stably transfected with TLR4 and an NF-κB reporter gene were transiently transfected with plasmids encoding for CD14 and either MD-2 (25 ng) or MD-2s (wedge represents 25–200 ng). Twenty-four hours later, cells were left untreated or stimulated with LPS (50 ng/ml) for 6 h. Mean relative stimulation of luciferase activity ± SD for a representative experiment, each performed in triplicate, is shown. D, Supernatants were also collected and measured for IL-8 secretion. Pearl Gray et al. J Immunol 2010;184:6359-6366 Copyright © 2010 by The American Association of Immunologists, Inc.