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. 2014 Feb 10;211(2):313–328. doi: 10.1084/jem.20122844

Figure 4.

Figure 4.

USP21 inhibits TRIM25- and RNF135-induced RIG-I activation. (A) HEK293T cells were transfected with indicated plasmids for 24 h, and then left uninfected or infected with SeV for 12 h before IFN-β luciferase activity was measured. (B) HEK293T cells were transfected with MYC-USP21 WT, CA mutant, MYC-CYLD, or MYC-A20 along TRIM25. Transfected cells were left uninfected or infected with SeV for 12 h before IFN-β luciferase activity was measured. (C) Expression vector encoding FLAG-RIG-I, RNF135, and HA-Ub were co-transfected with control, MYC-USP21 WT, CA mutant, MYC-CYLD, or MYC-A20. Cell lysates were immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-HA antibodies. (D) HEK293T cells were transfected with expression vectors encoding MYC-USP21 WT, CA mutant, MYC-CYLD, or MYC-A20. MYC-tagged proteins were immunoprecipitated with anti-MYC antibodies. In the parallel experiments, HEK293T cells were transfected with FLAG-RIG-I, RNF135, and HA-Ub. Supernatant containing polyubiquitinated FLAG-RIG-I proteins were incubated with immunoprecipitated MYC-USP21 WT, CA mutant, MYC-CYLD, or MYC-A20 for 2 h at 30°C, and then immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-HA antibodies. Error bars indicate ±SD in duplicate experiments. Data are representative of two (A and B) or three (C and D) independent experiments.