Anti hcv core

The primary mouse antibodies included anti-HCV core (Thermo, Waltham, MA, USA), anti-HCV NS5A (ViroGen Co, Watertown, MA, USA), anti-SRB1 antibody (BD, Franklin Lakes, NJ, USA), and anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO, USA). The primary rabbit antibody was anti-claudin-1 antibody (Abcam, Cambridge, MA, USA). Secondary antibodies included HRP-conjugated ECL goat anti-rabbit IgG, HRP-conjugated ECL goat anti-mouse IgG (Amersham Biosciences, Piscataway, NJ, USA), and goat anti-mouse IgG conjugated with AlexaFluor 594 (Invitrogen, Carlsbad, CA, USA).

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), followed by sonification. Proteins were separated by SDS-PAGE with 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, anti-STAT2, anti-phospho-STAT2, anti-MAVS, anti-TRIF, anti-PKR, anti-IRF3 (Cell Signaling Technology, Inc., Beverly, MA), anti-HCV core, (Thermoscientific), anti-HCV NS3, anti-HCV NS5A and anti-NS5B (Virogen, Watertown, MA), anti-ISG15, anti-MxA and anti-TRIF (Abcam), anti-TRF3 (Santa Cruz), anti-phospho-IRF3 (Origene) and anti-β-actin (Sigma Life Science and Biochemicals, St. Louis, MO). DENV-2 NS3 was detected by a specific monoclonal antibody against NS3 (Yao-Hong Biotechnology). 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 employed to detect chemiluminescent signals. Immunoblots shown in each figure are representative of three independent experiments. Densitometry was performed with ImageJ software. Student's t-test was used as statistical test.

Alcohol was purchased from Kelong Bio Inc. (Chengdu, China). Chloroquine (CQ), 3-methyladenine (3-MA), anti-P62/SQSTM1 antibody, and secondary antibodies for western blot (horseradish peroxidase-conjugated goat-anti-rabbit IgG, and goat-anti-mouse IgG) were purchased from Sigma-Aldrich China (Shanghai, China). Anti-β-actin, anti-ATG7, anti-ATG3 antibodies were purchased from Abcam Trading China (Shanghai, China). Anti-HCV core, anti-ATG5–ATG12, and Lipofectamine3000 were purchased from Thermo Fisher Scientific China (Shanghai, China). Anti-PIASy, anti-LC3B, anti-Beclin 1, anti-SUMO1, and anti-SUMO2 were purchased from Cell Signaling Technology China (Shanghai, China), and anti-CYP2E1 and anti-ADH were purchased from Novus Biologicals China (Shanghai, China).