FIG. 7.

Activation of TNF-α by MHV-A59 infection. (A) Schematic diagram showing the sequence elements of the murine TNF-α (mTNF-α) promoter and the binding sites for transcription factors NF-κB (κB), CRE, AP-1, and Sp1. (B) Nuclear translocation of NF-κB following MHV-A59 infection. Cells were infected with MHV-A59 at an MOI of 5 for 18 h (right) or mock infected (left). The cells were fixed with 4% paraformaldehyde and stained with primary anti-NF-κB p65 polyclonal antibody and fluorescein isothiocyanate-conjugated goat anti-rabbit antibody. The images were taken with a digital camera under a fluorescence microscope. The arrows indicate the nuclear translocation. (C) Assay for NF-κB activation. Primary astrocytes were infected with MHV-A59 at an MOI of 5 or mock infected. At 18 h p.i., nuclear extracts were isolated and NF-κB activity was determined with the TransAM NF-κB p65 kit as described in Materials and Methods. The results are expressed as the mean absorbance at 450 nm (OD₄₅₀) for three independent experiments. The error bars indicate standard deviations of the mean. The asterisk indicates statistical significance between the pair (P < 0.05).