Adenoviral-mediated gene transfer of A20 achieves high levels of expression in cultured PAEC. Cultured 90% to 100% confluent PAEC, from the fifth or the sixth passage, were either not infected (untreated) or infected with either the control rAd.β-gal (adeno-beta-gal) or rAd.A20 (adeno A20). Forty-eight hours following transduction, PAEC were recovered and cytospinned on glass slides using a cytospin 3 apparatus (Shandon Inc, Pittsburgh, PA) before being assessed by immunohistochemistry for the expression of the A20 transgene. Immunohistochemical analysis was performed using a mouse anti-human A20 MoAb of the IgG1 isotype, followed by a secondary peroxidase-conjugated goat anti-mouse antibody (Dako, Carpinteria, CA; right panel). A nonrelevant mouse MoAb was used as a control (left panel). Results show low to undetectable levels of A20 in noninfected or rAd.β-gal–infected PAEC. In contrast PAEC transduced with the rAd.A20 showed high levels of expression of A20 in >95% of the cells. Expression of the A20 protein was limited to the cytoplasm.