Journal: The Journal of Experimental Medicine
Article Title: Lipopolysaccharide Interaction with Cell Surface Toll-like Receptor 4-MD-2
Figure Lengend Snippet: E5531 acts on LPS interaction with TLR4-MD-2 at a concentration that does not affect LPS binding to mCD14. (a) Ba/F3 cells expressing TLR4-MD-2 and CD14 were pretreated with or without E5531 (indicated concentration) at 37°C for 30 min. Cells were stimulated with medium alone or 3 μg/ml LPS at 37°C for 30 min. After washing, cells were stained with biotinylated MTS510 mAb followed by streptavidin-PE (left and middle columns), or with anti-LPS followed by goat anti–mouse IgG-FITC (right column). Open histograms depict staining with the secondary reagent alone. (b) 3 μg/ml LPS with indicated concentrations of E5531 was added to the supernatant from Ba/F3 cells expressing CD14. sCD14 in the supernatant was precipitated with anti-CD14 mAb, followed by immunoprobing with anti-LPS (top) or anti-CD14 (bottom). (c) After treatment with E5531 and LPS as in panel a, cells were subjected to cell surface biotinylation, washing, detergent lysis, immunoprecipitation with Sa15-21, SDS-PAGE (polyacrylamide gel:18.0% for LPS and 7.5% for TLR4; under nonreducing conditions), and electroblotting. Precipitated LPS (top) and cell surface TLR4 (bottom) were probed with anti-LPS mAb or alkaline phosphatase–conjugated streptavidin, respectively. (d) After treatment with LPS and E5531 as in panel c, cells were subjected to detergent lysis, SDS-PAGE, electroblotting, and immunoprobing IkBα (top) or actin (bottom).
Article Snippet: Cells were washed in staining buffer, and incubated with R-PE–conjugated goat anti–rat IgG Ab (Southern Biotechnology Associates Inc.), FITC-conjugated goat anti–mouse IgG (BD Biosciences), or R-PE–conjugated streptavidin (BD Biosciences) for 15 min.
Techniques: Concentration Assay, Binding Assay, Expressing, Staining, Lysis, Immunoprecipitation, SDS Page