A1 expression is NF-κB–dependent. (A) HUVEC were stimulated with TNF (100 U/mL), LPS (100 ng/mL), or PMA (5 × 10⁻⁸ mol/L) for 3 hours. A1 or Bcl-X_L mRNA was detected using -[³²P]-dATP–labeled specific cDNA probes. (B) HUVEC were noninfected (NI) or infected with rAd.A20, rAd.IκB or rAd.β-gal. Forty-eight hours after infection, HUVEC were either left nontreated (−) or were stimulated with TNF (+) (100 U/mL). A1, A20, and IκB mRNA levels were detected by Northern blot analysis using their specific -[³²P]-dATP–labeled cDNA probes. GAPDH was used to confirm equal quantity of RNA. Result shown is representative of three independent experiments. (C) BAEC were either nontransfected (NT) or transfected with a control plasmid (pcDNA₃) or the p65 expression plasmid (+p65). A1 mRNA was only induced in p65 transfected BAEC (lane 5 v 3 and 4). Lanes 1 and 2 correspond to negative (C) and positive controls (TNF treated). GAPDH amplification (0.45 kb) was similar in all samples for all cycles tested. Only the 20 cycles’ amplification results are shown.