Figure 1 | Scientific Reports

Figure 1

From: A Hierarchical Mechanism of RIG-I Ubiquitination Provides Sensitivity, Robustness and Synergy in Antiviral Immune Responses

Figure 1

The double mutant of RIG-I markedly blocks the K63-linked ubiquitination of FL-RIG-I, whereas single mutants exhibit only small effects.

(A) Schematic of RIG-I-2CARD ubiquitination sites. (B) Luciferase assay of RIG-I-2CARD and different mutants. (C) Luciferase assay of full length RIG-I (FL-RIG-I) or its mutants. HEK 293T cells were transfected with wild type (WT) FL-RIG-I and its mutants, together with ISRE-luc, followed by intracellular (IC) poly (I:C) treatment for 24 hr. (D) HEK 293T cells were transfected with WT RIG-I or its indicated mutants, followed by IC poly (I:C) treatment for 24 hr. Cell lysates were collected for immunoblot analysis. (E) The RIG-I K172R/K164R double mutant (DM) markedly blocks K63-linked ubiquitination of RIG-I. HEK-293T cells transfected with wild type (WT) RIG-I or DM together with HA-K63-Ub were treated with or without IC poly (I:C) for 12 hr. Lysates were collected and immunoprecipitated with anti-Flag and immunoblotted with anti-HA. (F) HEK-293T cells were transfected with Flag/HA-RIG-I WT or DM. After treatment with IC poly (I:C) for 9 hr, the lysates were collected and immunoprecipitated with anti-Flag and immunoblotted with anti-HA.

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