A16104

Protein samples were collected via chemical cell lysis using RIPA buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 1% Triton X-100, 0.1% sodium deoxycholate, 140 mM NaCl, 0.1% SDS) and normalized by total protein concentration before adding SDS-PAGE sample buffer (Bio-Rad). Protein samples were loaded and run on a 4–20% polyacrylamide gels (Bio-Rad). Gels were transferred to nitrocellulose membranes before being blocked with PBS containing 5% (w/v) non-fat dried milk and 0.1% Tween-20 for at least 1 hour at room temperature or overnight at 4°C. Membranes were incubated with primary antibody diluted in PBS containing 5% (w/v) non-fat dried milk and 0.1% Tween-20 for at least 1 hour at room temperature or overnight at 4°C overnight. Primary antibodies used included anti-N1 (4A5, gift from Gene Tan at J. Craig Venter Institute), anti-NP (GeneTex GTX125989), anti-PB1 (GeneTex GTX125923), and anti-GAPDH (Abcam ab181603). Membranes were washed 3 times with PBS containing 0.1% Tween-20 before being incubated with anti-mouse-HRP (Invitrogen A16072) or anti-rabbit-HRP (Invitrogen A16104) secondary antibodies for 1 hour at room temperature. Membranes were washed 3 times with PBS containing 0.1% Tween-20 before treatment with Clarity or Clarity Max ECL (Bio-Rad) and exposure to film for development. Uncropped Western blots are shown in S10 Fig.