FIG 9.

TRIM21 promotes K27-linked ubiquitination of MAVS. (A) TRIM21 promotes the K27-linked ubiquitination of MAVS. HEK293T cells were cotransfected with the indicated plasmids for 42 h and treated with MG132 (25 μM; Sigma) for an additional 6 h. Ubiquitination and immunoblotting assays were performed with the indicated antibodies. (B) The RING domain of TRIM21 is essential for the K27-linked polyubiquitination of MAVS. HEK293T cells were cotransfected with the indicated plasmids for 42 h and treated with MG132 (25 μM) for an additional 6 h. Ubiquitination and immunoblotting assays were performed with the indicated antibodies. (C) Luciferase activity of lysates in HEK 293T cells transfected for 24 h with IFN-β–Luc or ISRE-Luc plus Flag-MAVS, along with V5-TRIM21-FL, V5-TRIM21-DR, V5-TRIM21-DPS, or an empty vector. (D and E) HLCZ01 cells were transfected with pSilencer-vector or pSilencer-TRIM21 for 24 h and then infected with NDV (D) or SeV (E) for 16 h. Ubiquitination and immunoblotting assays were performed with the indicated antibodies. (F) Huh7.5.1(MAVS-C508R) cells were transfected with pSilencer-vector or pSilencer-TRIM21 for 24 h and then infected with HCV (MOI = 2) for 72 h. Ubiquitination and immunoblotting assays were performed with the indicated antibodies. (G) HLCZ01-sh-con and HLCZ01-sh-IFNAR1 cell lines were transfected with pHA-ub (K27) plasmid for 36 h and then treated with IFN-α (200U/ml) for 6 h. Ubiquitination assays and immunoblotting were performed with the indicated antibodies. The results are presented as means ± standard deviations. *, P < 0.05; **, P < 0.01 versus control.