Fig. 4.

Suppression of poly (I:C)-induced IRF3 activation by protein 8b and 8ab. a. Suppression of IRF3 dimerization by proteins 8b and 8ab. Huh7 cells were transfected with 4 μg of empty vector, Flag-8b and Flag8ab, respectively, followed by stimulation with poly(I:C) for 20 h. Whole cell lysates were subjected to either native PAGE or SDS-PAGE and probed with anti-IRF3. Tubulin was included as a loading control. The ratio of dimeric IRF3 to monomeric IRF3 was calculated as the band intensity of monomer divided by the band intensity of dimer. b. Suppression of poly (I:C)-induced IFN-β promoter activity by proteins 8b and 8ab. Huh7 cells were transfected with control vector pcDNA3.1, pcDNA-8b and pcDNA-8ab, respectively, together with a luciferase reporter construct under the control of IFN-β promoter. At 24 h post-transfection, cells were then further transfected with poly (I:C). At 24 h post-stimulation, cells were lysed and measured for the firefly luciferase activity. pRL-TK was also co-transfected to serve as an internal control. Data were represented as mean of triplicates from 3 independent experiments. c-f. Huh7 cells were transfected with 4 μg of empty vector, Flag-8b and Flag8ab, respectively, followed by stimulation with poly(I:C) for 20 h. Total RNA was then extracted for quantitative real-time RT-PCR with specific primers for IFN−β (c), ISG56 (d), RANTES (e) and ISG15 (f). The expression of each gene was expressed relative to their respective control sample transfected with empty vector. Data were represented as mean of replicates from 2 independent experiments. GAPDH was used as internal control.