Hep c cag c7 50

Overnight, Huh-7.5 cells (1.5 × 10⁴ cells/well) seeded in a 96-well plate were inoculated for 3 h with HCVcc containing a supernatant at a MOI of 0.1. Next, the virus was removed, and the cells were overlaid with fresh medium with different concentrations of compounds or sofosbuvir. Three days post infection, the cells were washed with PBS, fixed with methanol for 30 min, and permeabilized in 0.5% Triton X100 in PBS for 5 min followed by another wash with PBS, and immunostaining (IPMA) to detect pseudo-plaques was performed. An anti-core antibody (Hep C cAg (C7-50); Santa Cruz Biotechnology, Dallas, TX, USA; 1:300 dilution) was used as the primary antibody, and anti-mouse HRP labeled antibody (1:1000 dilution) was used as the secondary antibody. HCV-positive pseudo-plaques were detected using the Vector Nova Red kit (Vector Laboratories Ltd., Peterborough, UK), and IC₅₀ was calculated as the concentration at which the number of pseudo-plaques (foci) was reduced by 50% compared to infected control cells using the GraphPad Prism software.

Cells were washed twice with phosphate‐buffered saline and harvested in whole‐cell extract lysis buffer (10 mm Tris/HCl pH 7.05, 50 mm NaCl, 1% (w/v) Triton X‐100, 0.5 mΜ PMSF and protease/phospho‐protease inhibitor cocktails by Roche). Protein concentrations were measured with the MicroBCA assay (Thermo Scientific). 40 μg of cell lysates were resolved in 10% (v/v) SDS/PAGE gels and transferred onto nitrocellulose membranes. After blocking, membranes were incubated overnight with primary antibodies. Membranes were then washed and incubated with the appropriate secondary antibody for 90 min at room temperature. Chemiluminescence was detected using Pierce ECL western blotting substrate (Thermo Scientific).
The following antibodies were used in this study: SMAD4 (#9515) by Cell Signaling (Danvers, Massachusetts, USA); TMPRSS6 (ab56180), by Abcam (Cambridge, UK); β‐actin (MAB1501) by Millipore (Burlington, Massachusetts, USA); ferroportin 1 (MTP11) by Alpha Diagnostics (San Antonio, Texas, USA); and STAT3 (sc‐483) and HepC Cag (C7‐50) by Santa Cruz Biotechnology (Dallas, Texas, USA). The polyclonal HCV NS5A and HCV‐1a core antibodies used in this study have been described before [43, 47].
Hepatitis C virus NS5A expression following induction of the cell clones was examined by immunofluorescence with the HCV NS5A polyclonal antibody, as previously described [41].