Figure 3.

The inhibition of T7-GFP expression by viperin is not at the translational level. (a) Viperin does not affect global protein synthesis. HEK293T cells were seeded in a 24-well plate. The next day the cells were transfected the indicated amounts of a viperin expressing plasmid and incubated for 48 h. The cells were then incubated at 37 °C in the presence of puromycin followed Western blotting with puromycin, viperin and tubulin specific antibodies. Cycloheximide (Chx) was used as a negative control. (b) Viperin does not inhibit cap or IRES dependent translation. HEK293-T7 cells were co-transfected with the indicated increasing amounts of viperin and a bicistronic plasmid expressing dsRed in a cap dependent manner and GFP from an IRES. The cells were lysed and analyzed by Western blot with viperin (red), GFP or RFP antibodies (green) 24 h post-transfection. (c) Viperin does not affect T7-polymerase protein levels. BHK-21 cells were seeded to 24-wells plate. The next day the cells were co-transfected with viperin or empty plasmid (50 ng/well) together with T7 polymerase and T7-GFP (400 ng/well). The cells we’re lysed and analyzed by Western blot 48 h post-transfection using T7 polymerase (green), tubulin (red), GFP (green) and viperin (red) antibodies. Full-length blots are presented in Supplementary Fig. 3.