A1 inhibits NF-κB reporter activity. (A) Inhibition is upstream of p65-mediated transactivation, (B) and is comparable to that achieved with Bcl-2; (C) A1 expression does not inhibit the NF-κB–independent reporter, HIV-▵κB CAT (D). (A) BAEC were transfected with the HA-A1 expression plasmid titrated (0, 1 ng, 10 ng, 0.1 μg, or 0.5 μg) with pcDNA₃ to equal 0.5 μg of DNA together with the NF-κB (0.7 μg) and β-gal (0.3 μg) reporters. Forty-eight hours after transfection, cells were stimulated with either TNF (100 U/mL) (lanes 6 through 10) or LPS (100 ng/mL) (lanes 11 through 15) for 7 hours. Data shown are representative of six experiments. (B) BAEC were cotransfected with 0.5 μg of pcDNA₃ or HA-A1 expression plasmid along with NF-κB reporter (0.7 μg), β-gal reporter (0.3 μg) and in the presence of increasing amounts of p65 expression plasmid ranging from 0 to 100 ng. Data shown represent the fold induction of the NF-κB reporter luciferase activity by increasing amount of cotransfected p65. Data shown are representative of three experiments performed. (C) BAEC were cotransfected with 0.5 μg of pAC or murine Bcl-2 expression plasmid along with NF-κB reporter (0.7 μg), β-gal reporter (0.3 μg), and increasing amounts of p65 expression plasmid ranging from 0 to 100 ng. Data shown represent the fold induction of the NF-κB reporter luciferase activity by increasing amount of cotransfected p65. Data shown are representative of two experiments performed. (D) BAEC were cotransfected with 0.5 μg of pcDNA₃ or HA-A1 together with 0.6 μg of an HIV-▵κB CAT reporter in the absence (−) or presence of 0.2 μg (+) of the viral protein c-Tat. Results are given in cpm ± SE. All error bars are ± SE. Data shown are representative of three experiments performed.