Ecl donkey anti rabbit igg

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).

Western blotting and protein detection were performed using standard protocols (30 (link)) in unstimulated Beas-2B cells cultured to confluence in 6-well plates or following siRNA transfection, as detailed later. Primary antibodies purchased from Santa Cruz included anti-AHR (sc-133088X), anti-NFKB p65 (sc-372), and anti-GAPDH (sc-25778). Secondary antibodies were ECL Donkey anti-rabbit IgG, HRP-linked F(ab')₂ fragment (NA9340) and ECL Sheep antimouse IgG, HRP-linked whole Ab (NA931), both from GE Healthcare/Amersham.