Anti pb2

Rabbit anti-PA, anti-PB1, anti-PB2, and anti-NP antibodies were purchased from GeneTex Inc. (GeneTex, United States). Mouse monoclonal antibodies against influenza A virus HA and M2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from Beyotime Biotechnology. The antibodies of human RNA pol II, RNA pol II CTD phosphor Ser2 and RNA pol II CTD phosphor Ser5 were purchased from Active Motif company.

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.