RIG-I expression in cell culture

Plasmid pEF-Bos-FLAG RIG-I MIII was a gift from Curt Horvath (Addgene plasmid # 52877; http://n2t.net/addgene:52877; RRID:Addgene_52877) which expresses RIG-I with a mutation to motif III [³³], was transiently expressed in HEK293-T. The cells were transfected with the plasmid and a negative control (empty vector) in 6-well plates using polyethyleneimine (PEI) [³⁴]. Cells were infected with SLEV at a MOI of 0.01 48 h after transfection and supernatants were collected at time points 24 h and 48 h post-infection to quantify the viral load. Expression of transfected FLAG-RIG-I was confirmed by anti-FLAG Western blot using a mouse monoclonal anti-FLAG (Sigma). Anti-actin mouse monoclonal antibody was purchased from Abcam.