Fig. 2.

Cloning and characterization of the −1 kb fragment of the MD-2 promoter using deletion mutants. Top panel demonstrates cloning of deletion mutants within the −1 kb fragment of the MD-2 gene promoter using primers flanked by the arrows. Bottom panel demonstrates transfection of T84 cells with −1 kb-MD-2 pGL3 or deletion mutants as indicated. The day following transfection, T84 cells were stimulated with IFN-γ and cells lysed for luciferase and β-galactosidase measurements. Reporter gene activation was significantly higher in cells transfected with −1 kb-MD-2 pGL3 or the mutants compared with the empty pGL3 vector control (data not shown). Stimulation with IFN-γ significantly increased −1 kb-MD-2 pGL3 reporter gene activation in T84 cells. This IFN-γ-dependent activation was abrogated with deletion of the −728 to −1 kb region. This graph is one experiment representative of three with similar findings and was performed in triplicate. Error bars indicate standard deviation.