A1 expression inhibits E-selectin, IL-8, and IκB reporter activity. (A) Western blot analysis of A1 expressing BAEC. A1 protein was detected using anti-HA antibody at the expected molecular weight of 20 kD, with as little as 0.001 μg (lane 2) of transfected HA-A1 expression plasmid and expression was near optimal with 0.1 μg (lane 4) (B) 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 E-selectin (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. Results are given in relative light units. Data shown are representative of six experiments. Error bars are ± SE (C and D) BAEC were transfected with 0.5 μg of the HA-A1 expression plasmid together with the IL-8 or IκB (0.7 μg) and β-gal (0.3 μg) reporters. BAEC were then stimulated with TNF (100 U/mL) (lanes 3 through 4) or LPS (100 ng/mL) (lanes 5 through 6). Results are given in relative light units. Data shown are representative of three experiments.