Anti ns1

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.