Investigating EcTRAF4 Regulation of IFN and NF-κB

To examine the effects of EcTRAF4 on the activity of IFN and the nuclear factor-κB (NF-κB) promoter, luciferase plasmids, including those encoding zebrafish IFN3–Luc, human IFN-stimulated response element (ISRE)–Luc, and NF-κB–Luc (Promega, Madison, WI, USA), were used [37 (link),40 (link)]. In brief, GS cells were co-transfected with 200 ng of plasmid-encoding ISRE–Luc, IFN3–Luc, or NF-κB-Luc and 600 ng of pcDNA3.1-3 × HA or plasmid-encoding HA–EcTRAF4. A total of 50 ng of pRL-SV40 Renilla luciferase vector (Promega, Madison, WI, USA) was used as the internal control. The cells were harvested after 48 h to measure luciferase activities with the Dual-Luciferase^® Reporter Assay System (Promega, Madison, WI, USA), according to the manufacturer’s instructions.