Figure 1. ISGylation is required for MDA5-, but not RIG-I, signaling.

(a, b) ELISA of IFN-β from supernatants of MEFs (WT or Isg15^−/−) (a) and HeLa cells (WT or ISG15 KO) (b) transiently transfected with increasing amounts of FLAG-tagged MDA5 or RIG-I for 40 h. Whole cell lysates (WCLs) were probed by immunoblotting (IB) with anti-ISG15, anti-FLAG, and anti-Actin (loading control). (c) ELISA of IFN-β from supernatants of WT or Isg15^−/− MEFs that were mock-stimulated or transfected with EMCV-RNA (0.1 or 0.4 μg/mL), HMW-poly (I:C) (0.5 μg/mL), or RABV_Le (1 pmol/mL), or infected with SeV (10 HAU/mL) for 24 h. (d) Quantitative RT-PCR (qRT-PCR) analysis of IFNB1 and CCL5 mRNA in WT and Isg15^−/− MEFs stimulated as in (c). (e) IRF3 phosphorylation in the WCLs of NHLFs that were transfected with the indicated siRNAs for 30 h and then mock-stimulated or transfected with EMCV-RNA (0.4 μg/mL) or RABV_Le (1 pmol/mL) for 6 h, assessed by IB with anti-pS396-IRF3 and anti-IRF3. (f) ELISA of IFN-β from supernatants of NHLFs that were transfected with the indicated siRNAs for 30 h and then mock-stimulated or transfected with EMCV-RNA (0.4 μg/mL) or RABV_Le (1 pmol/mL), or infected with SeV (10 HAU/mL) for 16 h. (g) ELISA of IFN-β from the supernatants of PBMCs that were transduced for 40 h with the indicated shRNAs and then infected with mutEMCV (MOI 10) or SeV (200 HAU/mL) for 8 h. (h) qRT-PCR analysis of IFNA2 and IL-6 mRNA in PBMCs that were transduced and infected as in (g). Data are representative of at least two independent experiments (mean ± s.d. of n = 3 biological replicates in a, b, c, d, f and mean of n = 2 biological replicates in g and h). *p < 0.05, **p < 0.01, ***p < 0.001 (unpaired Student’s t-test). ND, not detected; NS, not significant.