Figure 2

RelA nuclear levels are diminished in ORF73-expressing cells. (A) Reduction of NF-B binding to B sequences in ORF73-expressing cells correlates with diminished levels of nuclear RelA. HEK 293T cells were transiently transfected with an ORF73 expression plasmid, or control transfected. After 24 h of culture, cells were stimulated with TNF for 40 min, or left unstimulated. Nuclear extracts were prepared and subjected to electromobility shift assays (EMSAs) using the NF-B consensus oligonucleotide from the immunoglobulin promoter region. The percentage of NF-B binding present in each condition is shown. Nuclear extracts were analysed by immunoblotting to determine the input of RelA and ORF73 present in each binding reaction. Densitometry analysis of RelA nuclear levels present in each experimental condition, normalised to the nuclear protein LaminB, is shown. (B) ORF73 protein does not bind to B sequences. Nuclear extracts from HEK 293T cells expressing ORF73 were subjected to a supershift assay using the specified antibodies. (C) Immunofluorescence analysis of ORF73-expressing cells. HEK 293T cells were stimulated with 50 ng/ml of TNF for the times indicated and then subjected to immunostaining with anti-RelA and anti-ORF73 antibodies. -, without; +, with; , anti; WB, western blotting.