Quantification of Immune Signaling Proteins

The levels of tyrosine-phosphorylated STAT1, RIG-I, IFN regulatory factor 3 (IRF3), and phospho-IRF3 were measured by Western blot analysis as described previously [22 (link),46 (link)]. Whole cell lysates were prepared from virus-infected Calu-3 cultures (MOI = 1) at the indicated time points. Proteins were resolved by electrophoresis on 8% polyacrylamide Tris-Glycine gels (Invitrogen, Carlsbad, CA, USA), and then transferred to polyvinylidene difluoride (PVDF) membranes. The levels of tyrosine-phosphorylated STAT1, RIG-I, IRF3, and phospho-IRF3 were then measured by immunoblotting with mouse monoclonal anti-phospho-Y⁷⁰¹-STAT1 Ab, rabbit monoclonal anti-RIG-I Ab, rabbit monoclonal anti-IRF3 Ab, and rabbit monoclonal phospho-IRF3, respectively (Cell Signaling Technology, Beverly, MA, USA). The levels of viral PB1 and M1 proteins in H1N1 viruses carrying silent PB1 and M1 mutations were analyzed by western blot with rabbit anti-PB1 (ThermoFisher Scientific, Rockford, IL, USA) and mouse monoclonal anti-M1 Abs (Abcam, Cambridge, MA, USA), respectively.