Supplementary Figure 3: ELF4 is involved in the antiviral immune signaling.

(a&b) 293T cells were transfected with the empty vector (EV), MAVS or increasing amounts of ELF4 and infected with VSV-GFP or WNV, 24 h later, the viral loads were analyzed by microscopy (a) and using a plaque assay (b). **P<0.01 as determined by Student's t-test. Data were pooled from 3 independent experiments. There are 2 technical replicates within each experiment. (c) 293T cells were transfected with empty vector (EV), increasing amounts of Flag-ELF4 or Flag-STK38 and infected with SeV. Lysates were immunoblotted with antibodies to SeV, Flag or GAPDH. Data were pooled from 3 independent experiments. Data were pooled from 3 independent experiments. (d) 293T cells were transfected with the empty vector (EV), scrambled or ELF4 specific siRNA. 48 h later, lysates were immunoblotted using antibodies to ELF4 or GAPDH. (e) 293T cells were transfected with increasing amounts of scrambled or ELF4 specific siRNA and infected with VSV-GFP, 24 hours later, the VSV-GFP was analyzed by FACS. *P < 0.05 as determined by Student's t-test. Data were pooled from 3 independent experiments. There are 2 technical replicates within each experiment. (f) Confirmation of ELF4 knockout by immune blotting. Data were pooled from 3 independent experiments. (g) Wild type, Sting^Gt/Gtor Elf4^-/-peritoneal macrophages were infected with VSV-GFP. 24 or 48 h later, the viral loads were analyzed by FACS. **P<0.01 as determined by Student's t-test. Data were pooled from 3 independent experiments. There are 2 technical replicates within each experiment. (h) Elf4^-/- MEF cells were transfected with wild type ELF4 or the mutant lacking ETS domain. 24 hours later, the cells along with wild type MEF and untransfected cells were infected with VSV-GFP. Data were pooled from 3 independent experiments. (i&j) Development of specific IgG and IgM against WNV. Serum was collected from wild-type or Elf4-/- mice at the indicated days after infection. The levels of specific IgG were determined by incubating serum with adsorbed control or purified WNV E protein. Data are the mean of serum from 8 mice per time point performed in duplicate. * P<0.05 (nonparametric Mann-Whitney analysis).Data were pooled from 3 independent experiments. (k&l) Cxcl10 and IL-6 mRNA (relative to β-actin expression) in whole blood from mice at days 0, 1 and 3 after infection with 200 PFU of WNV subcutaneously. Data were pooled from 3 independent experiments. (m) NK cells and NK1.1+CD3+ NKT cells were sorted from splenocytes of WT mice. 1X10⁶ NK cells and 1X10⁶ NKT cells were injected intravenously (i.v.) into recipient mice. Mortality of age- and sex-matched wild-type (WT; C57BL/6) and Elf4-/-mice inoculated subcutaneously (s.c.; n = 10 mice per group) with 200 PFU of WNV and monitored daily for 15 d. p<0.0001 using the Log-rank test. Data were pooled from 2 independent experiments.(n&o) Proportion of CD4+ T cells and CD8+T cells in spleen of mice at day 0, or day6 after subcutaneous infection with 200 PFU of WNV (s.c.; n = 8 mice per group), assessed by FACS. Data were pooled from 3 independent experiments. No randomization or blinding was used in the animal experiments.