Anti np

Total protein was lysed using RIPA buffer and normalized. Protein samples were separated by 10% SDS-PAGE and then transferred onto PVDF membranes (Roche, Switzerland). The protein membranes were incubated at 4°C overnight with primary antibodies at the appropriate concentrations, followed by incubation with an anti-mouse/rabbit secondary antibody labeled with horseradish peroxidase (Cell Signaling Technology, Danvers, MA, USA; dilution 1:10,000) for 1 h. The protein bands were visualized using a FluorChem E imaging system (ProteinSimple, San Jose, CA, USA). The primary antibodies were used: anti-PB2 (GeneTex, Irvine, CA, USA; dilution 1:1,000), anti-NP (GeneTex; dilution 1:1,000), anti-TRAF3 (Cell Signaling Technology; dilution 1:1,000), anti-IRF3 (Cell Signaling Technology; dilution 1:1,000), anti-HA-UB (Cell Signaling Technology; dilution 1:1,000), and anti-p-IRF3 (Ser386) (Bioss, Shanghai, China; dilution 1:1,000). GAPDH or β-actin was used as a protein control.

Cells were harvested in lysis buffer (50 mM Tris pH 8.0, 5 mM NaCl, 0.5% NP-40, and 1X protease inhibitor), frozen and thawed three times, and then the proteins were recovered. Immunoprecipitation (IP) proceeded overnight at 4 °C in IP buffer containing antibodies against Grail or Flag. The IP mixture was then incubated with Dynabeads Protein G (Invitrogen) for 1 h prior to isolation using a DynaMag magnet and washing three times with SNNTE buffer (5% sucrose, 1% NP-40, 0.5 M NaCl, 50 mM Tris pH 7.4, and 5 mM EDTA). The immunoprecipitates were resuspended in SDS-PAGE sample buffer, boiled, and loaded onto a gel. Following separation, the proteins were transferred to a nitrocellulose membrane and the blot was probed with antibodies diluted in PBS/Tween 20 with 5% non-fat milk. Antibody detection was carried out using enhanced chemiluminescence reagents (GE Healthcare), as described by the manufacturer. The primary antibodies used for immunoblotting were: anti-PA (GeneTex), anti-PB1 (GeneTex), anti-PB2 (GeneTex), anti-HA (GeneTex), anti-NA (GeneTex), anti-NP (GeneTex), anti-M1 (GeneTex), anti-M2 (GeneTex), anti-NS1 (GeneTex), anti-HA (81B8, Cell Signaling, USA), anti-beta actin (MAb1501, Chemicon), and anti-Grail antibodies.

MDCK cells in 6-well plates were infected with 1.5 × 10³ TCID50 of the H1N1 influenza virus and treated with NC-5 (10 μM, 40 μM, and 80 μM) and OC (10 μM). Cells were lysed after 24 h. In a time-addition experiment, the MDCK cells were infected with virus at a multiplicity of infection (MOI) of 5, and co-incubated for 1 h at 4 °C. After changing the supernatant, the cells were cultured at 37 °C. NC-5 (80 μM) was added at distinct time-points, as described in Results. Ten hours post-infection, cell lysates were harvested. The following antibodies were applied for immunoblotting: anti-M1 (Catalog No. GTX125928, GeneTex), anti-NP (Catalog No. GTX83054, GeneTex), and anti-β-actin (Catalog No. SC-47778, Santa Cruz). A Bio-Imaging system was applied for band intensity detection, and the proteins were quantified using ImageJ, which was normalized to β-actin levels.

pHW181-PB2, pHW182-PB1, pHW183-PA, pHW185-NP and PolI-luc plasmids are derived from influenza A/WSN/33 virus [24 (link)]. Rabbit anti-PB1, anti-PB2, anti-PA, anti-NP, and mouse anti-M1, anti-GAPDH, anti-p84 antibodies were purchased from GeneTex (Irvine, CA, USA). Mouse anti-β-actin antibody was purchased from Abcam (Cambridge, MA, USA).