Supporting information for Mullen et al. (2003) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0630495100
Fig. 7.
MD-2 supernatants that lack monomer are active. (A) Human embryonic kidney (HEK) 293T and HeLa cells were transfected with WT MD-2 in the presence of 10 % FBS, and after 16 h media were replaced with either serum-free medium or medium supplemented with 10% FBS. Supernatants were collected 24 h later, immunoprecipitated, and analyzed by SDS/PAGE under nonreducing conditions. Exposures are shown at 30 s (Left) and 5 min (Right) for HEK293T cells and at 10 s (Left) and 1 min (Right) for HeLa cells. Arrows indicate monomeric MD-2. (B) TLR4 reporter cells were treated with lipopolysaccharide and the MD-2 supernatants shown in A. The serum free samples were supplemented with 10% FBS prior to experimentation as a source of sCD14. NF-k B activities are presented as luciferase units. Mock indicates supernatants from mock-transfected HEK293T cells.