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. 2018 May 25;8:8100. doi: 10.1038/s41598-018-26516-z

Figure 1.

Figure 1

Viperin inhibits T7-GFP expression. (a) Viperin is the only ISG specifically inhibiting expression from T7-GFP. HEK293-T7 cells were infected with ISG expressing lentiviruses. After 24 h the cells were co-transfected with T7-GFP and a CFP-YFP fusion protein expressed from a CMV promoter. The cells were analyzed 48 h post-transfection by Western blot with a GFP antibody. RFP expression was used to confirm lentivirus infection. Actin was used as a loading control. Bands showing the effect of viperin on T7-GFP are labeled with white arrow head. Quantification of the bands compared to luciferase control and normalized to actin is shown at the top. (b) Viperin inhibits T7-GFP expression in a dose dependent manner. HEK293-T7 cells were transfected with the indicated amounts of viperin-expressing plasmid. After 24 h the cells were transfected with a T7-GFP or CMV-GFP. The cells were lysed after 48 h and analyzed by Western blot using GFP, tubulin and viperin antibodies. Quantification was performed as described above and is shown at the bottom of each panel. Presented is a representative gel from at least 3 independent repeats. (c) Viperin inhibits T7-GFP expression. HEK293-T7 cells were transfected with viperin (50 ng/well). The next day the cells were transfected with T7-GFP. Images were obtained 48 h after transfection. Bar = 100 µM. Presented are representative images from at least 5 independent repeats. (d) Viperin inhibits T7-Luciferase expression. HEK293-T7 cells were transfected with the indicated amounts of viperin. The next day the cells were co-transfected with T7-Firefly luciferase (0.8 µg/well) and ß-galactosidase (10 ng/well) expressing plasmids. The cells were lysed 48 h post-transfection and analyzed for luciferase and ß-galactosidase activity. Presented is representative graph from at least 5 independent repeats preformed in triplicates. (e) Viperin inhibits T7-GFP expression from ectopically expressed T7 polymerase. HEK293T cells were co-transfected with increasing amounts of viperin together with T7-GFP and T7 polymerase. The cells were lysed and analyzed by Western blot with GFP (green) and viperin (red) specific antibodies 24 h post-transfection. Tubulin (red, top) was used as a loading control. Full-length blots are presented in Supplementary Fig. 1.