FIG. 4.
Effect of EZ infection on binding to the ISRE, GAS, and NF-κB elements. HUVECs were either mock infected or EZ infected at an MOI of 1.0 for 75 h and treated with either IFN-α, IFN-γ, or IL-1β for the final 3 h. Gel shift analysis was performed as previously described (17, 33) with nuclear extracts by using either the ISRE from the ISG54 gene (A), the GAS element from the IRF-1 gene (B), or the NF-κB binding site from the IL-6 promoter (C) as a probe. Both the specific and nonspecific competitor DNAs [IL-6 NF-κB site in panel A, the mouse immunoglobulin kappa light chain gene NF-κB site in panel B, and (IRF-E)2 in panel C] were added in 30-fold excess and tested by using the mock IFN-α sample in panel A, mock IFN-γ sample in panel B, and mock-infected sample treated with 100 μg of poly(I-C) per ml (Pharmacia Biotech, Piscataway, N.J.) for 6 h in panel C. Supershift analysis (B) was performed with a monoclonal antibody (Ab) to the N-terminal region of STAT-1α. Binding complexes were resolved by 4% nondenaturing polyacrylamide gel electrophoresis and visualized by autoradiography (17, 33).