Mouse monoclonal anti hcv core antibody

For NS3 immunostaining, cells were seeded in four-well chamber slides (Nunc), washed with phosphate-buffered saline (PBS) (Hyclone), and fixed with 3.7% formaldehyde for 10 min at room temperature. Cells were permeabilized with 0.1% Triton X-100 in PBS buffer for 5 min at room temperature, and then blocked with 1% bovine serum albumin (BSA; Affymetrix, Cleveland, OH) in PBS buffer for 20 min at room temperature. After washing with PBS, cells were incubated with mouse monoclonal anti-HCV-Core antibody (Thermo Scientific, Grand Island, NY) diluted 1:300 in PBS or anti-HCV-NS3 antibody (Thermo Scientific) diluted 1:50 in PBS for 1 h at room temperature. Slides were washed with PBS and mounted using VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA). Images were visualized on an LSM-700 confocal microscope (Carl Zeiss, Jena, Germany).

Mouse monoclonal anti-HCV core antibody and rapamycin were purchased from Thermo Fisher Scientific (Rockford, IL, USA). Mouse monoclonal anti-SREBP-1c, mouse monoclonal anti-fatty acid synthase (FAS), and goat polyclonal anti-stearoyl CoA desaturase (SCD) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal anti-HCV NS5A antibody was obtained from Virogen (Watertown, MA, USA). Polyclonal antibodies specific to mTOR, lipin-1, and acetyl CoA carboxylase (ACACA) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Scrambled miRNA, miR-99a-5p mimics, and miR-99a-5p inhibitor were synthesized and obtained from Genolution Pharmaceuticals (Seoul, Korea). We used miR-99a-5p because miR-99a-5p is a dominant arm among the two strands of miRNA duplex. Scrambled small interfering RNA (siRNA) and siRNA targeting mTOR were obtained from Cell Signaling Technology, Inc. Cells were transfected with miRNA mimics or siRNAs using lipofectamine^TM 2000 (Thermo-Fisher Scientific, MA, USA), according to the manufacturer’s instructions at a final concentration of 50 nM. pcDNA3_flag_mTOR and pcDNA3.1_flag_SREBP-1c were purchased from Addgene (Cambridge, MA, USA).

Mouse monoclonal anti-GRIM-19 antibody was purchased from Abcam (Cambridge, MA, USA). Mouse monoclonal anti-β-actin and mouse monoclonal anti-flag antibodies were obtained from Sigma–Aldrich (St. Louis, MO, USA). Mouse monoclonal anti-HCV core antibody was purchased from Thermo Scientific (Rockford, IL, USA). Mouse monoclonal anti-HCV NS5A antibody was obtained from Virogen (Watertown, MA, USA). Polyclonal antibodies specific to phospho-STAT3, STAT3 (Ser-705), and acetyl CoA carboxylase (ACC) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated anti-mouse, anti-rabbit immunoglobulin G (IgG), mouse monoclonal anti-SREBP-1c, mouse monoclonal anti-fatty acid synthase (FAS), and goat polyclonal anti-stearoyl CoA desaturase (SCD) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Scrambled siRNA and siRNA targeting GRIM-19 were obtained from Santa Cruz Biotechnology.