(A) Northern blot analysis of E-selectin, VCAM-1, IL-8, and IκBα gene induction. PAEC were noninfected (NI), rAd.A20, and rAd.β-gal–infected at a MOI of 500 as in Fig 1. Forty-eight hours following infection, PAEC were either left nontreated (C) or were stimulated with TNF (T) (100 U/mL), LPS (L) (100 ng/mL), or PMA (P) (5.10⁻⁸ mmol/L). A20, E-selectin, VCAM-1, IL-8, IκBα, and GAPDH steady-state transcript levels were quantitated in these samples by Northern blot analysis using α-[³²P]–dATP–labeled homologous or cross-reactive cDNA probes as described in Materials and Methods. Results show that A20 expression in PAEC (confirmed by Northern analysis for A20 mRNA) significantly inhibits E-selectin, VCAM-1, and IκBα gene induction (>80% to 90%) following stimulation by TNF, LPS, and PMA as compared with high level of induction in either NI cells or rAd.β-gal–infected cells. A20 expression also achieved significant inhibition (>70%) of IL-8 gene induction. Results shown are representative of three independent experiments. (B) Inhibition of cell-surface expression of the EC-specific adhesion molecule E-selectin. Confluent PAEC in 96-well microtiter plates were infected as in (A). Triplicate wells of PAEC were either untreated (C) or treated with the same stimuli as in (A) extended to α-thrombin (Th) and H₂O₂ (H) (300 μmol/L). Expression of the E-selectin protein was analyzed by ELISA 4 hours following stimulation. Results confirm and extend to α-thrombin and oxidative stimuli, the previous mRNA results, by showing that A20 abrogates surface-expression of E-selectin in PAEC for all stimuli tested. Results shown are representative of three independent experiments.