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Figure 2 | Virology Journal

Figure 2

From: Identification of an effective siRNA target site and functional regulatory elements, within the hepatitis B virus posttranscriptional regulatory element

Figure 2

The effect of siRNA expression plasmids on reporter activity. (A) ShRNA template oligonucleotides of siRNA anti PRE 1317-1337 and PRE 1329-1349. (B) The effects of pShRNA plasmids on luciferase activity. The line graphs indicate normalized luciferase activities of transfected COS-7 cells with pSilencer- Negative, pSilencer-GAPDH, pShRNA PRE1317-1337 and pShRNA 1329-1349. COS-7 cells were transfected in triplicate with 95 ng of pLucSplice/fPRE, 5 ng of phRL-SV40 and various amount of pSiRNA expression plasmids as indicated, pUC18 was used to make up the total DNA to 1 μg. Cells were harvested at day 1, day 2 and day 3 post-transfection and analyzed for expression of luciferase protein. '***' indicates significant differences of normalized luciferase activities compared to 0 ng of siRNA expression plasmid with p < 0.001 (by t-test). (C) The effect of pShRNA PRE1317-1337 on the luciferase activity in HuH-7 cells. In this experiment, cells were transiently transfected in triplicate with 45 ng of the pSpliceLuc/fPRE, 5 ng of the phRL-SV40 and different amounts of the pShRNA constructs as indicated. pSilencer-Negative was used to make up total DNA plasmid to 350 ng. Cells were harvested and analyzed luciferase activity and western blot analysis after 48 h of incubation. Reporter expression levels are higher from these SV40 promoter containing constructs in COS- 7 cells (B) than in Huh-7 cells (C). The bar graph shows normalized luciferase activity of mean values of three independent experiments; error bars represent standard deviation. '***' indicates significant differences of normalized luciferase activities comparing to the control siRNA expression plasmid (pSilencer-GAPDH) with p < 0.001 (by t-test). Western blot analysis with primary antibodies: anti-GAPDH, anti-firefly luciferase protein (anti-Luc) and anti-Poly A binding protein (anti-PABP) and the horseradish peroxidase-conjugated secondary antibody.

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