Gammabind g plus sepharose

For transient-transfection and coimmunoprecipitation experiments, HEK293T cells (1 × 10⁶) were transfected with the respective plasmids for 24 h and then were lysed in 1 ml of lysis buffer (20 mM Tris [pH 7.5] 150 mM NaCl, 1% Triton-X, 1 mM EDTA, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). For each immunoprecipitation, a 0.4-ml aliquot of lysate was incubated with 0.2 μg of the indicated antibodies or control IgG and 25 μl of 1:1 slurry of Gammabind G plus Sepharose (Amersham Biosciences) for 2 h. The Sepharose beads were washed three times with 1 ml of lysis buffer containing 500 mM NaCl. The precipitates were analyzed by Western blotting as previously described (38 (link)).
For endogenous coimmunoprecipitation experiments, PK-15 cells (5 × 10⁷) were infected with FMDV (multiplicity of infection [MOI], 1.0) for the indicated time. Cells were then lysed in 5 ml lysis buffer and the lysate was incubated with 1 μl of the indicated antiserum or preimmune control serum. The subsequent procedures were carried out as described above.

For transient-transfection and Co-IP experiments, HEK293T cells (2 × 10⁶) were transfected with the respective plasmids for 24 h. Then, they were lysed in 1 mL of lysis buffer [20 mM Tris (pH 7.5) 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10 µg/mL aprotinin, 10 µg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride]. For each immunoprecipitation, a 0.4 mL aliquot of lysate was incubated with 0.2 µg of the indicated antibodies or control IgG and 25 µL of 1:1 slurry of Gammabind G plus Sepharose (Amersham Biosciences) for 2 h. Sepharose beads were washed thrice with 1 mL of lysis buffer containing 500 mM NaCl. The precipitates were analyzed by western blotting as previously described (45 (link)). For endogenous Co-IP experiments, GAMs (2 × 10⁷) were infected with PPRV for the indicated periods. Co-IP and immunoblotting were performed as previously described.

PK-15 (porcine kidney cells) and HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). We used mouse monoclonal antibodies against Flag, β-actin (Sigma, St. Louis, MO, USA), hemagglutinin (HA) (Covance), Myc, green fluorescent protein (GFP), and TBK1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Rabbit polyclonal antibodies against IRF3 and phosphorylated IRF3 were also employed (Santa Cruz Biotechnology, Santa Cruz, CA, USA). IFN-γ (PeproTech, Rocky Hill, NJ, USA) was used for IFN stimulation. Gamma Bind G Plus-Sepharose was purchased from Amersham Biosciences and M-MLV reverse transcriptase was from Invitrogen. The Sendai virus (SeV) used in the experiments and rabbit antibodies against VISA, MDA5, TRAF3, and RIG-I were previously described44 (link)45 (link)46 (link). The type O FMDV was propagated in PK-15 cells, and the supernatants of infected cells were clarified and stored at −80 °C.