Journal: Journal of Virology

Article Title: Phosphorylation of Serine 225 in Hepatitis C Virus NS5A Regulates Protein-Protein Interactions

doi: 10.1128/jvi.00805-17

Figure Lengend Snippet: Figure 6. Effects of siRNA ablation of NS5A interacting protein expression on 627

Article Snippet: Huh7 cells stably harbouring a wildtype SGR were untransfected, or transfected with pooled siRNA targeting VAP-A, VAP-B, or a control scrambled siRNA (Santa Cruz) at a final concentration of 10 nM.

Techniques: Expressing

Journal: Journal of Virology

Article Title: Phosphorylation of Serine 225 in Hepatitis C Virus NS5A Regulates Protein-Protein Interactions

doi: 10.1128/jvi.00805-17

Figure Lengend Snippet: Figure 8. Effects of siRNA ablation of VAP-A or VAP-B on the distribution of 652

Article Snippet: Huh7 cells stably harbouring a wildtype SGR were untransfected, or transfected with pooled siRNA targeting VAP-A, VAP-B, or a control scrambled siRNA (Santa Cruz) at a final concentration of 10 nM.

Techniques:

Journal: Journal of Virology

Article Title: Phosphorylation of Serine 225 in Hepatitis C Virus NS5A Regulates Protein-Protein Interactions

doi: 10.1128/jvi.00805-17

Figure Lengend Snippet: Figure 6. Effects of siRNA ablation of NS5A interacting protein expression on NS5A expression and RNA replication. (a) Huh7 cells stably harbouring a wild-type SGR were transfected with pooled siRNA targeting NAP1L1, Bin1, VAP-A or VAP-B, or a control scrambled siRNA (Santa Cruz) at a final concentration of 10 nM. Transfected cells were incubated in medium lacking G418, lysed at 72 hpt and analysed by Western blotting. (b) Total NS5A levels were quantified from fluorescent Western blots (n=3). The Western blots shown are representative of three independent experiments. Asterisks indicate a significant difference from the control value ****(P<0.0001). (c) At 72 hpt, the cells were harvested in TRIzol, total RNA was extracted and qRT-PCR was conducted on 100 ng total cellular RNA using 5′-UTR TaqMan primers (31). The data shown are from three independent experiments. Significant differences denoted by **** (P<0.0001). (d) Huh7 cells stably harbouring the S225A mutant SGR were transfected with indicated siRNA and analysed by Western blotting at 72 hpt as described in (a).

Article Snippet: Huh7 cells stably harbouring a wildtype SGR were untransfected, or transfected with pooled siRNA targeting VAP-A, VAP-B, or a control scrambled siRNA (Santa Cruz) at a final concentration of 10 nM.

Techniques: Expressing, Stable Transfection, Transfection, Control, Concentration Assay, Incubation, Western Blot, Quantitative RT-PCR, Mutagenesis

Journal: Journal of Virology

Article Title: Phosphorylation of Serine 225 in Hepatitis C Virus NS5A Regulates Protein-Protein Interactions

doi: 10.1128/jvi.00805-17

Figure Lengend Snippet: Figure 7. Effects of siRNA ablation of NAP1L1 or Bin1 on the distribution of NS5A. Huh7 cells stably harbouring a wild-type SGR were untransfected, or transfected with pooled siRNA targeting NAP1L1, Bin1, or a control scrambled siRNA (Santa Cruz) at a final concentration of 10 nM. At 72 hpt, cells were permeabilised, immunostained for NS5A (green panels), NAP1L1 (a, red panel), or Bin1 (b, red panel) and imaged by confocal microscopy. Scale bar, 20 µm and 5 µm. Spatial data for NS5A were determined from 12 cells for each assay using the ImageJ software package. The data shown are representative of three independent experiments. Asterisks indicate a significant difference from the control value (****P < 0.0001).

Article Snippet: Huh7 cells stably harbouring a wildtype SGR were untransfected, or transfected with pooled siRNA targeting VAP-A, VAP-B, or a control scrambled siRNA (Santa Cruz) at a final concentration of 10 nM.

Techniques: Stable Transfection, Transfection, Control, Concentration Assay, Confocal Microscopy, Software

Journal: Journal of Virology

Article Title: Phosphorylation of Serine 225 in Hepatitis C Virus NS5A Regulates Protein-Protein Interactions

doi: 10.1128/jvi.00805-17

Figure Lengend Snippet: Figure 8. Effects of siRNA ablation of VAP-A or VAP-B on the distribution of NS5A. Huh7 cells stably harbouring a wildtype SGR were untransfected, or transfected with pooled siRNA targeting VAP-A, VAP-B, or a control scrambled siRNA (Santa Cruz) at a final concentration of 10 nM. At 72 hpt, cells were permeabilised, immunostained for NS5A (green panels), VAP-A (a, red panel), or VAP-B (b, red panel) and imaged by confocal microscopy. Scale bar, 20 µm and 5 µm. Spatial data for NS5A were determined from 12 cells for each assay using the ImageJ software package. The data shown are representative of three independent experiments. Asterisks indicate a significant difference from the control value (***P < 0.001).

Article Snippet: Huh7 cells stably harbouring a wildtype SGR were untransfected, or transfected with pooled siRNA targeting VAP-A, VAP-B, or a control scrambled siRNA (Santa Cruz) at a final concentration of 10 nM.

Techniques: Stable Transfection, Transfection, Control, Concentration Assay, Confocal Microscopy, Software

Journal: Journal of Virology

Article Title: Phosphorylation of Serine 225 in Hepatitis C Virus NS5A Regulates Protein-Protein Interactions

doi: 10.1128/jvi.00805-17

Figure Lengend Snippet: Figure 9. Effects of siRNA ablation of NAP1L1, Bin1 or VAP-A on the distribution of NS5A in the absence of genome replication. Huh7-Lunet T7 cells were untransfected, or transfected with pooled siRNA targeting NAP1L1, Bin1, VAP-A, or a control scrambled siRNA (Santa Cruz) at a final concentration of 10 nM. At 48 hpt, cells were transfected with pTM-NS3-5B plasmid. After a further 24 h, cells were permeabilised, immunostained for NS5A (green panels), NAP1L1 (a, red panel), Bin1 (b, red panel) or VAP-A (c, red panel) and imaged by confocal microscopy. Scale bar, 20 μm and 5 μm. Spatial data for NS5A were determined from 20 cells for each assay using the Image J software package. Asterisks indicate a significant difference from the control value (****P < 0.0001).

Article Snippet: Huh7 cells stably harbouring a wildtype SGR were untransfected, or transfected with pooled siRNA targeting VAP-A, VAP-B, or a control scrambled siRNA (Santa Cruz) at a final concentration of 10 nM.

Techniques: Transfection, Control, Concentration Assay, Plasmid Preparation, Confocal Microscopy, Software