Ag281

In total, 10 μg of each cell lysate sample was analysed by 10 % SDS-PAGE. The proteins were transferred onto a polyvinylidene difluoride member using a Trans-Blot Turbo system (Bio-Rad, Hercules, CA, USA). The membrane was blocked for 1 h using 5 % nonfat milk in TBST buffer (50 mmol l⁻¹ Tris base, 150 mmol l⁻¹ NaCl, pH 7.5, 0.05 % Tween 20). The membrane was incubated with anti-GFP, anti-gB, anti-Cas9 and anti-β-actin antibodies (anti-GFP=1 : 1000, AG281, Beyotime; anti-gB=1 : 12 500, ab6506, Abcam; anti-β-actin=1 : 12 500, A1978, Sigma-Aldrich; anti-Cas9=1 : 1000, ab191468, Abcam) for 1 h after blocking. After washing five times, the membrane was incubated with HRP-conjugated goat anti-mouse antibody (1 : 10 000, Sigma-Aldrich) for 40 min and then washed three times with TBST buffer. Signals were visualized using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare).

Protein samples obtained from cell lysates were mixed with 5× protein loading buffer, boiled at 100 °C for 10 min and then subjected to SDS-PAGE. Proteins were separated and transferred to a PVDF membrane (Roche). The nonspecific antibody binding sites were blocked with 5% skim milk in PBS-Tween (PBST). Membranes were incubated with mouse anti-EGFP (AG281, Beyotime) (1: 2000) or mouse anti-tubulin (AT819, Beyotime) (1: 2000) primary antibodies diluted in PBS at 4 °C overnight, and then incubated with HRP-conjugated goat anti-mouse IgG secondary antibody diluted in PBS (1: 1000) at 37 °C for 1 h. The signals were detected using an ECL imaging system. The images were obtained from a CanoScan LiDE 100 scanner (Canon) and quantities of protein blots were measured with Image J software.

The following antibodies were used: anti-HCV core antibody (C7-50, Abcam), anti-HCV NS3 antibody (H23, Abcam), monoclonal mouse GFP antibody (AG281, Beyotime), monoclonal mouse anti-Flag antibody (F1804, Sigma), monoclonal mouse HA antibody (AH158, Beyotime), polyclonal rabbit HA Antibody (Sc-805, Santa Cruz), anti-DENV NS1 antibody (ab41616, Abcam), anti-β-actin antibody (BioVision), anti-CIDEB monoclonal antibody (M01, Abnova), anti-ApoE antibody (10817-RP02, Sino Biological), polyclonal anti-CIDEB antibody (LS-C119539, Lifespan), goat anti-mouse antibody (Chemicon), goat anti-rabbit antibody (Chemicon) conjugated with horseradish peroxidase, Cy3-labeled goat anti-mouse IgG (H + L) (Beyotime), Alexa Fluor 647-labeled goat anti-rabbit IgG (Beyotime), Alexa Fluor 555-labeled donkey anti-rabbit IgG (H + L) (Beyotime) and Alexa Fluor 488-labeled donkey anti-mouse IgG (H + L) (Invitrogen).
The siRNA sequences specifically targeting HCV, CD81, PI4KIIIα, ApoE and RACK1 were previously reported18 (link)47 (link). The target sequences of the CIDEB siRNA were as follows: 5′-AGAGGAGGATGGAACTGCA-3′ (siCIDEB 213) and 5′-AGTACTCAGGGAGCTCC-3′ (siCIDEB 517).