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. 2017 Jan 31;91(4):e01138-16. doi: 10.1128/JVI.01138-16

FIG 4.

FIG 4

LUBAC inhibits LMP1-promoted IRF7 transcriptional activity. (A) 293 cells in 24-well plates were transfected with 150 ng LUBAC (equal amounts of each component), 10 ng Flag-LMP1, 50 ng Myc-IRF7, 40 ng pGL3/IFN-β–Luc, and 10 ng Renilla luciferase. A dual-luciferase assay was performed 24 h after transfection. (B) 293 cells in 24-well plates were transfected with 150 ng LUBAC (equal amounts of each component), 10 ng Flag-LMP1, and 50 ng IRF7 or IRF3. IFN-β production in the medium was measured 48 h after transfection with a human IFN-β enzyme-linked immunosorbent assay (ELISA) kit following the manufacturer's instructions (PBL Assay Science). (C) A20.2J (Rnf31+/+) and H294.10 (Rnf31−/−) cells were transfected with 1 μg HA-LMP1, 2 μg Myc-IRF7, 1 μg pGL3/IFN-β–Luc, and 0.5 μg Renilla luciferase using a Nucleofector kit. Dual-luciferase assays and immunoblotting were performed after 24 h. Experiments were repeated at least three times, and representative results are shown.