Fig. 3.

Functional characterization of components of the B/NS1 interactome. (a) Western blot analysis of specific B/NS1 interactors during the course of FLUBV (B/Yamagata/88) infection in A549s (m.o.i. of 5 p.f.u. cell⁻¹). (b) Western blot analysis of A549 cell lysates previously treated or not with rIFNα (1000 IU ml⁻¹, 20 h). The indicated proteins were detected with specific antibodies. (c–e) Reporter assay for IFNβ-promoter induction. A549-pr(IFNb).GFP cells were transfected for 48 h with specific siRNAs before IFNβ-promoter activation was induced by SeV infection (c). The number of GFP-positive cells was monitored over 24 h, and normalized to overall cell confluency. The area under the curve (AUC) in relation to the untreated control from three independent experiments is depicted (d, green bars, left axis: error bars=sd). Dotted line indicates four sd values away from the negative control. In addition, cell viability after siRNA transfection was measured, and mean relative light units (RLU) from three independent experiments are depicted in relation to the untreated sample (d, black lines, right axis: error bars=sd). Dashed line indicates 80 % cell viability. (e) Heat map representation of the data shown in (d), indicating levels of IFNβ-promoter activation. (f) Western blot analysis of 293 T cells co-transfected (or mock) for 48 h with a plasmid expressing FLAG-ILF3 and the indicated siRNAs. (g) A549 cells were transfected with the indicated siRNAs for 48 h before infection with FLUBV (B/Yamagata/88) at an m.o.i. of 1 p.f.u. cell⁻¹. Bars represent mean viral titres in supernatants at 48 h post-infection (n=2, error bars represent sd).