Riplet function is dispensable for RIG-I RNA binding activity.

<p>(A) Expression vectors encoding Riplet, FLAG-tagged RIG-I CARDs, and/or FLAG-tagged RIG-I RD were transfected into HEK293FT cells together with an HA-tagged ubiquitin expression vector. Cell lysate was prepared at 24 hours after transfection, followed by immunoprecipitation with an anti-FLAG (RIG-I) antibody. (B) HEK293FT cells were transfected with expression vectors encoding FLAG-tagged RIG-I, Riplet, and HA-tagged ubiquitin. Cell lysate was prepared at 24 hours after transfection, and then incubated with biotin-conjugated (Biotin-dsRNA) or non-conjugated (dsRNA) double-stranded RNA. Biotin-dsRNA was pull-downed with streptavidin beads. Samples were subjected to SDS-PAGE, and proteins were detected by western blotting. (C) HEK293FT cells were transfected with FLAG-tagged wild-type RIG-I, RIG-I 5KR, or RIG-I K788R expression vector. 24 hours after the transfection, the cell lysate was prepared. The pull down assay with biotin-dsRNA was performed as described above.