Figure 5A SDS-PAGE analysis of proteins immunoprecipitated from A549 cell lysates using HA-antibody. Bands were stained by Coomassie Blue. Results were confirmed in three independent experiments, and representative data are shown. B Venn diagrams classifying the proteins identified in immunoprecipitated samples. A total of 318 proteins were identified by LC-MS/MS analysis. C The list of top ten networks obtained from Ingenuity Pathway Analysis of the 1918 PB1-F2 interactome. D The "viral infection" network of interactome proteins. Proteins shaded in gray directly interact with 1918 PB1-F2. Interferon signaling pathway is shown in yellow. Solid lines, protein-protein interactions; solid arrows, phosphorylation; dotted arrows, induction. E Analysis of interaction between DDX3 and 1918 PB1-F2. Cells were transfected with indicated plasmids; 18 h later, cells were treated with MG132 for 6 h. Cell lysates were immunoprecipitated with anti-HA antibody. Proteins were detected by immunoblotting with the indicated antibodies (left panel). IAV-infected cell lysates were immunoprecipitated with anti-PB1-F2 antibody and immunoblotted with the indicated antibodies (middle panel). Purified recombinant 1918 PB1-F2 protein was incubated with recombinant LysRS or LysRS-DDX3, and the interaction was analyzed by immunoblotting (right panel). Results were confirmed in three independent experiments, and representative data are shown. F Analysis of interaction between DDX3 and PB1-F2 mutants. A549 cells were transfected with the indicated clones; 18 h after transfection, cells were treated with MG132 for 6 h and analyzed by immunoprecipitation. Results were confirmed in three independent experiments, and representative data are shown. G A549 cells were treated with IFNβ for the indicated time periods and subjected to Western blotting (top). A549 cells were infected with indicated IAV. At 24 h post-infection, cell lysates were analyzed by Western blotting (bottom). Results were confirmed in three independent experiments, and representative data are shown. H Representative fluorescence images showing expression level of GFP-DDX3 (green). A549 cells were co-transfected with plasmids encoding the GEP-DDX3 and Flag-tagged PR8 or 1918 PB1-F2. At 18 h after transfection, cells were treated with MG132 for 6 h and subjected to fluorescence analysis. Results were confirmed in three independent experiments, and representative data are shown. I Mice were infected with 1×105 pfu of each virus, and lung homogenates were examined for endogenous Ddx3 expression. Results were confirmed in three independent experiments, and representative data are shown. J A549 cells were transfected with the indicated plasmids or treated with poly I:C. Cytoplasmic and nuclear fractions were separated and subjected to Western blotting. Results were confirmed in three independent experiments, and representative data are shown. K A549 cells were transfected with the indicated plasmids, and IFNβ mRNA levels were determined by semi-quantitative RT-PCR (top) and qPCR (middle). IFNβ promoter activity was determined by luciferase reporter assay (bottom). Results were confirmed in three independent experiments, and representative data are shown.