Mouse anti β actin monoclonal antibody

The primary antibodies used for this study were as follows: the mouse anti-HCV NS5A monoclonal antibody [H26] (Abcam, USA), mouse anti-HCV NS3 monoclonal antibody (Abcam, USA), goat anti-HCV core polyclonal antibody (Santa Cruz, USA), and mouse anti-β-actin monoclonal antibody (Boster, China). DAPI (Santa Cruz, USA) and Lipofectamine 2000 (Invitrogen, USA) was used per the manufacturer’s instructions.

After the indicated treatments, total cell extracts were obtained and lysed by RIPA buffer (KeyGENBioTECH, Nanjing, China). Protein concentrations were determined according to BCA Protein Assay Kit (Beyotime, Shanghai, China). The extracted proteins in the cell lysates were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The primary antibodies were as follows: rabbit anti-EGFR monoclonal antibody (Cell Signaling technology, Danvers, MA, USA), anti-PTEN monoclonal antibody (Cell Signaling technology, Danvers, MA, USA), anti-p-AKT(Ser473) monoclonal antibody (Cell Signaling technology, Danvers, MA, USA), anti-AKT monoclonal antibody (Cell Signaling technology, Danvers, MA, USA), anti-cyclin D1 monoclonal antibody (Santa Cruz Biotechnology, Dallas, USA), anti-Bcl2 monoclonal antibody (Santa Cruz Biotechnology, Dallas, USA), anti-Bax monoclonal antibody (Santa Cruz Biotechnology, Dallas, USA), anti-CYP2E1 monoclonal antibody (Epitomics, CA, USA) and mouse anti-β-actin monoclonal antibody (BOSTER, Wuhan, China). Secondary antibodies include HRP-Conjugated AffiniPure Goat Anti-rabbit IgG (ZSGB-BIO, Beijing, China). Immunoreactive proteins were visualized using ECL western blotting detection regents (GE Health-care, Buckinghamshire, UK).

Cells were lysed in sample buffer [62.5 mmol/L Tris-HCl pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate (SDS)] and boiled for 5 minutes. Equal amounts of lysate (50 μg total protein) were electrophoretically separated on 10% SDS/polyacrylamide gels and transferred onto nitrocellulose filter membranes (PALL, Boston, MA, USA, 66485) followed by incubation with a 1:1000-diluted anti-PLP2 antibody (Abcam, ab180131), anti-AKT phosphorylation (p-AKT) (CST, Danvers, MA, USA, 13038), anti-AKT (CST, 4685), anti-p21 (CST, 2947), anti-cyclin D (CST, 2978), and anti-GFP antibodies (CST, 2956). Proteins were detected by incubation with horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin G (1:3000) and enhanced by chemiluminescence (Pierce, Rockford, IL, USA, 32106). The membranes were stripped and reprobed with an anti-β-actin mouse monoclonal antibody (1:1000; Boster, Wuhan, CN, BM0627), which was used as a loading control.