Figure 3
Experimental validation of the hierarchical model of RIG-I ubiquitination.
(A) HEK-293T cells were transfected with wildtype (WT) RIG-I or the RIG-I K172R/K164R double mutant (DM) together with K63-ubiquitin (Ub) with or without IsoT, followed by intracellular (IC) poly (I:C) treatment for 12 hr. The lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-HA. (B) Intensity quantification of RIG-I ubiquitination levels in (A). (C) HEK 293T cells were transfected with RIG-I WT, K164R or K172R together with K63-Ub, with or without IsoT, followed by IC poly (I:C) treatment for 12 hr. The lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-HA. (D) Intensity quantification of RIG-I ubiquitination levels in (B). (E) HEK-293T cells were transfected with RIG-I WT, K164R, K172R or DM, together with K63-Ub, followed by IC poly (I:C) treatment for 12 hr. The lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-HA. (F) Intensity quantification of RIG-I ubiquitination level in (E). (G) Knockout efficiency of RIG-I knockout (KO) HEK-293T cells. (H) RIG-I KO cells were transfected with FL-RIG-I or its mutants together with ISRE-luc, followed by IC poly (I:C) treatment. (I) Interpretation of how the experimental results support hierarchical mechanism 1 and exclude the other models.
