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Fig. 5 Figure 5. Role of TRIM25 in the Perturbation of RIG-I-Mediated Antiviral Immunity under Sμg Conditions (A) IP analysis of ubiquitinated RIG-I in embryos microinjected with wild-type Ub-HA and FLAG-RIG-I, with or without poly(I:C) stimulation under Ng or Sμg conditions. The relative grayscale value of anti-HA was normalized to anti-FLAG within each treatment group. The data in the Ng control group were arbitrarily set to 1. (B) Overexpression of TRIM25 rescue the decrease of ubiquitination of RIG-I under Sμg conditions. Embryos were co-microinjected with wild-type Ub-HA and FLAG-RIG-I, with or without poly(I:C) stimulation under Ng or Sμg conditions, together with TRIM25-Myc or vector control. WCLs were used for IP and IB as indicated. Both Ng (lane 1) and Ng-pIC (lane 2) groups are used as controls. The former is used for comparison with Sμg (lane 3) and Sμg+TRIM25 groups (lane 4). The latter is used for comparison with Sμg-pIC (lane 5) and Sμg-pIC+TRIM25 groups (lane 6). The relative grayscale value of anti-HA was normalized to that of anti-FLAG within each treatment group. The data in the Ng control group were arbitrarily set to 1. (C) Analysis of IFNφ1 expression, IFNφ1, and NF-κB luciferase reporter assay in the indicated treatments with overexpression of TRIM25. (D) qPCR analysis of endogenous TRIM25 expression after IFNφ1-Myc induction and TRIM25MO-2 interference under Ng or Sμg conditions. (E) qPCR analysis of endogenous TRIM25 expression with or without poly(I:C) stimulation under Ng or Sμg conditions, as well as overexpression of IFNφ1-Myc. Error bars: All data points in this figure are presented as mean ± SE. Two-tailed unpaired t test. ∗∗p < 0.01; ∗p < 0.05.