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Figure 1 | Journal of Negative Results in BioMedicine

Figure 1

From: Refractoriness of hepatitis C virus internal ribosome entry site to processing by Dicer in vivo

Figure 1

miRNA-guided RNA silencing is not perturbed in cells harboring a subgenomic HCV replicon. (A) Schematic representation of the experimental strategy and reporter gene constructs. (B) HCV RNA expression in Huh-7 or 9–13 cells harbouring a subgenomic HCV replicon, treated or not with 100 IU/ml of interferon α-2B (IFNα-2B), was documented by Northern blot using a DNA probe complementary to HCV Internal ribosome entry site (nt 1 to 341). GAPDH was used as a loading control. (C) HCV NS3 and NS5B protein expression Huh-7 or 9–13 cells, treated or not with 100 IU/ml of IFNα-2B, was documented by Western blot using anti-NS3 1B6 (first panel) and anti-NS5B 5B-3B1 (third panel) antibodies, respectively. Actin was used as a loading control (second and fourth panels). (D) Huh-7 or 9–13 cells, treated or not with 100 IU/ml of IFNα-2B, were cotransfected using Lipofectamine 2000 with a Rluc:miRNA binding site construct, in which the Rluc reporter gene is coupled with 1 or 3 copies of perfectly complementary (PC) or natural wild-type (WT) binding sites (BS) for miR-328 (250 ng DNA), and a psiSTRIKE-based, pre-miR-328 expression construct (250 ng DNA). psiSTRIKE-Neg, which encodes a shRNA directed against a sequence deleted in the Rluc reporter mRNA, was used as a control. Results of Rluc activity were normalized with Fluc activity and expressed as a percentage of Rluc activity obtained with psiSTRIKE-Neg. Results are expressed as mean ± s.e.m. (n = 3 experiments, in duplicate).

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