Anti trif antibody

The cells were lysed in buffer containing 10 mM Tris-buffer (pH 7.6), 1% Triton X-100, 1% phosphatase inhibitor cocktail and 1 mM PMSF. The lysates were boiled in sodium dodecyl sulfate (SDS) sample buffer and were subjected to SDS–PAGE. Cytoplasmic extracts and nuclear extracts were obtained using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Sigma, 78833). Rabbit monoclonal antibodies against GAPDH, NLRP3, and ASC were purchased from Santa Cruz Biotechnology (CA, USA) and were diluted 1:1000. The anti-ATG5 antibody, anti-LC-3 antibody, anti-Caspase-1 antibody, anti-PCNA, and anti-p65 antibody were purchased from Cell Signaling Technology (Beverly, MA, USA) and were diluted 1:1000. The anti-TRIF antibody was purchased from Abcam and was diluted 1:1000. Horseradish peroxidase- conjugated goat anti-rabbit immunoglobulin G (Cell Signaling Technology, MA, USA) was used as the secondary antibody. Immunoreactive bands were identified using the ECL Western Blotting Detection System (Millipore Corporation, Billerica, MA, USA).

Cells were either mock-treated, LE-PolyI:C-treated, or infected with IAV viruses at a MOI of 0.1. At 6 h post-infection, the cells were washed with cold PBS and lysed for 15 min on ice with RIPA lysis buffer containing 50 mM Tris-HCl (pH7.4), 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate and a protease inhibitor cocktail (Beyotime Institute of Biotechnology, Beijing, China). Lysed cells were pelleted and the supernatants were incubated with the indicated antibodies (anti-TLR3 antibody or an isotype IgG antibody from Abcam) for 2 h at 4°C with gentle shaking. The samples were then added to a preprocessed EZview^TM Red Protein A/G Affinity Gel (Sigma) and incubated for 6 h. After washing three times with lysis buffer, the beads were boiled in SDS loading buffer, and then analyzed by immunoblot with the indicated antibodies (anti-TLR3 antibody or anti-TRIF antibody from Abcam).