Riplet affects RIG-I RD autorepression of CARDs signaling.

<p>(A, B) IFN-β promoter activation was determined using a p125luc IFN-β reporter gene. Expression vectors encoding RIG-I CARDs, full-length RIG-I, RIG-I K788R, RD, RD-K788, and/or Riplet were transfected into HEK293 cells as indicated. Reporter activation was determined after transfection. Data are presented as mean ± SD (n = 3). *p<0.05. (C) IFN-β promoter activation was determined using a p125luc IFN-β reporter gene. RIG-I and Riplet expression vectors were transfected into HEK293 cells as indicated. 24 hours after transfection, cells were infected with SeV at MOI = 1 for 24 hours, and the reporter activities were determined. (D–F) HEK293FT cells were transfected with expression vectors encoding FLAG-tagged RIG-I, HA-tagged TRIM25, HA-tagged Riplet, FLAG-tagged RIG-I K788R as indicated. Cell lysate was prepared at 24 hours after transfection, followed by immunoprecipitation with an anti-FLAG antibody (RIG-I). Relative band intensity of immunoprecipitated TRIM25 in panel D was determined (E). (G) Mouse splenocyte was isolated from wild-type and Riplet KO mice spleen. The cells were infected with SeV, and then cell lysate was prepared. Immunoprecipitation was carried out with anti-RIG-I rabbit mAb (D14G6), and subjected to SDS-PAGE. Endogenous K63-linked polyubiquitin chain was detected using K63-linked polyubiquitin chain specific antibody.