UL76 activates the NF-kB canonical pathway.

<p>A) Luciferase activity was determined in 293T cells co-transfected with pcDNA3 or pcDNA3HA-UL76, IL-8 luciferase reporter and either dominant negative IKKβ or IkBα (S32/36A) plasmids. Values of luciferase activity were normalized to β-Galactosidase activity. Data are expressed as means ± SD of triplicate wells from one of three similar experiments. Expression of UL76 and dominant negatives IKKβ or IkBα (S32/36A) was confirmed by western blot (below) using an anti-HA HRP-conjugated antibody. Detection of tubulin was used as loading control. B) Indirect immunofluorescence was performed on HFF cells transfected with pcDNA3HA-UL76 or control plasmid using an anti-p65 antibody and anti-mouse Texas Red conjugated secondary antibody. UL76 expression was detected using an anti-HA-FITC conjugated antibody and DAPI staining was used to define nuclei. Images were acquired with a 100× objective. C) Expression of p65 in HFF cells transfected with pcDNA3HA-UL76 or control plasmid was evaluated by western blot at the indicated time points. Detection of β-actin was used as loading control. As positive control cells were stimulated with TNFα. Levels of p65 relative to the levels of β-actin in the corresponding extracts were quantified by densitometry analysis and the fold levels relative to the control vector transfected cells are indicated below. D) 293T cells transfected with pcDNA3HA-UL76 or control plasmid were lysed for nuclear extraction at the indicated time points post-transfection. Nuclear extracts were immunoblotted with anti-p65, anti-β-actin and anti-nucleolin antibodies. Expression of UL76 was detected using an anti-HA antibody. Levels of p65 relative to the levels of nucleolin in the corresponding nuclear extracts were quantified by densitometry analysis. The fold levels relative to the control vector transfected cells are indicated below. E) Binding of NF-kB p65 subunit to the IL-8 promoter sequence (−121 to +61) or to a control region (−1042 to −826) in 293T cells expressing pcDNA3HA-UL76 or control plasmid was analyzed by ChIP assay at 30 h post-transfection using an anti-p65 antibody. Immunoprecipitated DNA was analyzed by quantitative real-time PCR. Data are expressed as fold enrichment relative to an unrelated IgG antibody used as control to nonspecific immunocomplexes.