Human umbilical vein endothelial cells (HUVECs) express functional dnIκB protein upon AddnIκB infection as demonstrated by western blot analysis and gene expression analysis by real time RT-PCR. (a) HUVECs were incubated with AddnIκB for 90 minutes at 37°C, in the absence or presence of 20 mg/ml recombinant viral knob, as described in Materials and methods. Cells were subsequently washed and incubated for another 24 hours. After preparation of cellular protein homogenate, western blotting was performed to detect IκB total protein (upper panel), the hemagglutinin-tagged transgene dnIκB (middle panel), and actin to control for protein loading (lower panel). (b) Non-infected (solid bar) and AddnIκB (open bar) or AdLacZ (gray bar) transduced HUVECs were activated with tumor necrosis factor (TNF)-α (100 ng/ml) for 24 h before real time RT-PCR was performed on mRNA isolated from each respective HUVEC incubation. Data were normalized to untreated, non-activated control HUVECs arbitrarily set at 1. Results are expressed as the mean ± standard deviation (n = 3). Asterisks indicate p < 0.05 compared with respective control cells without activation with TNF-α (TNF-α (-)). ICAM, intercellular adhesion molecule; ns, not significant; VCAM, vascular cell adhesion molecule; VEGF, vascular endothelial growth factor.