(A) Inhibition of NF-κB nuclear DNA-binding activity following TNF stimulation in rAd.A20–infected PAEC. Nuclear extracts were prepared from noninfected (NI), rAd.A20-infected, or rAd.β-gal–infected PAEC before and 2 hours following treatment with TNF(T) (100 U/mL) as described in Materials and Methods. NF-κB activation and binding to a specific κB binding oligo derived from the porcine IκBα promoter was evaluated by means of EMSA as described. Results reveal that nuclear extracts from PAEC expressing A20 had almost no inducible binding activity for NF-κB binding sites. Specificity of DNA binding was tested by the use of excess cold wild-type as a specific competitor (wt) or a mutant κB probe (mutκB) used as a nonspecific competitor. Results shown are representative of three independent experiments. (B) Western blot analysis of IκBα expression following TNF treatment. Cytoplasmic extracts from NI, rAd.A20, and rAd.β-gal–infected PAEC were recovered before and 10 minutes and 2 hours following TNF treatment and assessed for IκBα expression by means of Western blot analysis. Results show that A20 expression in PAEC inhibits the usual IκBα degradation that occurs 10 minutes following TNF stimulation. A second faster-migrating band is detected at 2 hours in the A20-expressing EC. Results shown are representative of three independent experiments.