Supplementary Figure 6: ELF4 is important for NF-κB– and ISRE-mediated transcription.

(a) 293T cells were transfected with empty vector (EV), ELF4 or MAVS and IFNβ-Luc, NFκB-Luc or AP1-Luc plasmid. *P<0.05 and ***P<0.001 as determined by Student's t-test. Data were pooled from 3 independent experiments. There are 2 technical replicates within each experiment. (b) Knockdown efficiency of p65 siRNA was analyzed in 293T cells by immunoblotting. Data were pooled from 3 independent experiments. (c&f) 293T cells were co-transfected with p65 or IRF3 and empty vector (EV) or increasing amounts of ELF4 and IFNβ-Luc (c) or NFκB-Luc (f) plasmid. ***P<0.001 as determined by Student's t-test. Data were pooled from 3 independent experiments. There are 2 technical replicates within each experiment. (d) 93T cells were transfected with Flag tagged ELF4 and HA tagged p50, p65 or IRF3. Cell lysates were immunoprecipitated with Flag and blotted with HA or Flag antibodies. WCL were blotted with Flag or HA antibodies. Data were pooled from 3 independent experiments.(e) MEF cells were transfected with scrambled siRNA or p65 specific siRNA. 36 hours later, cells were infected with SeV for 6 hours followed by chromatin immunoprecipitation. The cell lysates were immunoprecipitated by mouse IgG, or anti-ELF4, followed by qRT-PCR to quantify the IFNα2 and IFNα4 promoter DNA. **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. (g&i) 293T cells were co-transfected with IRF7 and empty vector (EV) or increasing amounts of ELF4 and IFNα2-Luc (g) or ISRE-Luc (i) plasmid. **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) 293T cells were co-transfected with IRF3 and empty vector (EV) or increasing amounts of ELF4 and ISRE-Luc plasmid. Data were pooled from 3 independent experiments. There are 2 technical replicates within each experiment. (j) In vitro expressed IRF3, p65 or ELF4 was incubated with the biotinylated IFNβ promoter probes followed by EMSA assay. Biotinylated probes were detected by HRP-streptavidin. Free probe (P), Shifted probe (S). Data were pooled from 3 independent experiments.