Mouse anti hcv core

Protein samples were prepared using RIPA buffer containing a protease inhibitor cocktail (Sigma Life Science and Biochemicals, St. Louis, MO, USA). Proteins were separated by SDS-PAGE and transferred to a PVDF membrane as previously described [3 (link)]. Primary antibodies included Rabbit anti-human ATG5 (Cell Signaling technology, Beverly, MA, USA), mouse anti-HCV core (Fisher Scientific, Pittsburgh, PA, USA), and mouse anti-β-actin (Sigma, St. Louis, MO, USA). The secondary antibodies were horseradish peroxidase (HRP)-conjugated enhanced chemiluminescence (ECL) donkey anti-rabbit IgG and HRP-conjugated ECL sheep anti-mouse IgG (GE Healthcare Biosciences, Pittsburgh, PA, USA). The blots were subjected to chemiluminescence assay using the Amersham ECL Western blotting detection kit (GE Healthcare Biosciences, Pittsburgh, PA, USA).

Cells were washed with PBS and lysed by use of RIPA buffer containing a protease inhibitor cocktail (Sigma Life Science and Biochemicals, St. Louis, MO). For HBV protein preparation, samples were heated at 56°C for 30 min. We loaded equal quantities of protein (20 μg) in all lanes. Protein samples were separated by SDS-PAGE with NuPAGE Novex precast 4 to 12% Bis-Tris gradient gels (Invitrogen, Carlsbad, CA) and blotted onto nitrocellulose membranes. The membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline with Tween 20 (TBST). Primary antibodies included mouse anti-human PKLR (Santa Cruz Biotechnology, TX), mouse anti-HCV core (Fisher Scientific, Pittsburgh, PA), mouse anti-HBcAg (Abcam, Cambridge, MA), and mouse anti-β-actin (Sigma, St. Louis, MO). The secondary antibodies were horseradish peroxidase (HRP)-conjugated enhanced chemiluminescence (ECL) donkey anti-rabbit IgG and HRP-conjugated ECL sheep anti-mouse IgG (GE Healthcare Biosciences, Pittsburgh, PA). The blots were subjected to chemiluminescence assay by use of an Amersham ECL Western blotting detection kit (GE Healthcare Biosciences, Pittsburgh, PA).

Cells were lysed using radioimmune precipitation assay (RIPA) buffer containing 1% NP-40, 0.1% SDS, 10 mM Tris–HCl (pH 7.4), 1 mM EDTA, 150 mM NaCl and protease inhibitor cocktail (Roche), and sonification was performed subsequently. Proteins were separated by SDS–PAGE or NuPAGE Novex pre-cast 4–12% Bis–Tris gradient gels (Invitrogen, Carlsbad, CA) and transferred to PVDF membranes. The primary antibodies used were: anti-STAT1, anti-phospho-STAT1 (Tyr701) anti-STAT2 and anti-phospho-STAT2 (Cell Signaling Technology, Inc., Beverly, MA), mouse anti-HCV core, (Thermoscientific), and mouse anti-actin (Sigma Life Science and Biochemicals, St. Louis, MO). Secondary antibodies were: HRP-conjugated ECL donkey anti-rabbit IgG and HRP-conjugated ECL sheep anti-mouse IgG (Amersham Biosciences, Piscataway, NJ). The ECL Western Blotting Detection Kit (Amersham Biosciences, Piscataway, NJ) was used to detect the chemiluminescent signals.