Different Subtypes of Influenza Viruses Target Different Human Proteins and Pathways Leading to Different Pathogenic Phenotypes
H5N1 NA and H1N1 NA have different impacts on IL-1β-induced NF-κB activation. 293 T cells in 24-well plates were cotransfected with 125 ng pNF-κB-luc, 25 ng pRL-TK and indicated amount of H5N1 NA (a) and H1N1 NA (b) expression plasmid, or empty vector for 24 h. Cells were then mock-treated or treated with IL-1β (10 ng/ml) for 6 h. Reporter activity was determined by dual-luciferase reporter assays. The resultant ratios were normalized to the fold-change value by that of IL-1β-untreated cells cotransfected with empty vector, pNF-κB-luc and pRL-TK. Data shown represent three independent experiments, with each determination performed in duplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (∗∗, Student’s t-test). 293 T cells were transfected with Flag-H5N1 NA (c) or Flag-H1N1 NA(d) or either HA-TAK1 or HA-TAB1 or HA-TAB2 or HA-IKKα or HA-KKβ or HA-vector expression plasmids for 30 h. All cells were then treated with IL-1β (10 ng/ml) for 30 min. The cells were lysed and subjected to immunoprecipitation (IP) using the mouse anti-HA tag. IP products and 5% input samples were analyzed by immunoblotting.
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