Figure 4
Involvement of TRIM25 in the regulation of MAVS. (A) HeLa cells were transfected with empty or TRIM25-V5 vector and co-transfected with an IFN-β promoter reporter as well as with renilla luciferase as an internal control. Twenty-four hours later, cells were transfected or not with HMW Poly(I:C) (1 μg/ml). Luciferase assay was performed 8 hr after transfection and was normalized using renilla luciferase activity. Data represent means ± SD (n = 3). (B) HeLa cells were transfected with control, MAVS, RIG-I and TRIM25 siRNAs for 48 hr, next transfected with an IFN-β promoter reporter as well as with renilla luciferase as an internal control. Twenty-four hours later, cells were transfected or not with Poly(I:C) (1 μg/ml). Luciferase assays were performed 8 hr after transfection and normalized using renilla luciferase activity. Data represent means ± SD (n = 3). The knockdown efficiency of MDA-5 and RIG-I was evaluated by immunoblot. For the knockdown of MAVS and TRIM25, see Figures 1D and 4E respectively. (C) Concentrations of mouse IFN-β in cellular supernatant from WT or TRIM25-/- MEFs, 9 hr after transfection with 2 μg/ml of Poly(I: C). IFN-β concentrations were assessed by ELISA. Data represent means ± SD (n = 2). (D) Concentrations of mouse IL-6 in cellular supernatant from WT or TRIM25-/- MEFs, 9 hr after transfection with 2 μg/ml of Poly(I: C). IL-6 concentrations were assessed by ELISA. Data represent means ± SD (n = 2). (E) HeLa cells were transfected with control or TRIM25 siRNAs for 72 hr. Then, cells were transfected or not with Poly(I:C) (1 μg/ml), and 9 hr after transfection TRIM25, MAVS, p-IRF3, IRF3, p-IκBα and IκBα were analyzed in cell extracts by immunoblotting. Actin was used as a protein loading control. Arrow indicates the phosphorylated state of MAVS. Values represent the ratio of the larger MAVS isoform band normalized with respect to the loading control. (F) WT or TRIM25-/- MEFs were transfected or not with Poly(I:C) (2 μg/ml), and 10 hr after transfection TRIM25, MAVS, p-IRF3, IRF3, p-IκBα and IκBα were analyzed in cell extracts by immunoblotting. Actin was used as a protein loading control. (F) WT or TRIM25-/- MEFs were transfected or not with Poly(I:C) (2 μg/ml) in the presence of MG132. Three hours later, MAVS and its ubiquitination were analyzed in mitochondrial extracts by immunoblotting with a short and a long exposure respectively. VDAC was used as a protein loading control.