Transient Transfection and Co-IP Assays in HEK293T Cells

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.