Pvdf membrane trans blot turbo transfer system

Cultured cells were lysed with RIPA cell lysis and extraction buffer (ThermoFisher) supplemented with Halt^TM Protease and Phosphatase Inhibitor Cocktail (ThermoFisher). Protein concentrations were measured using BCA Protein Assay kit (Solarbio). Protein concentrations were normalized with 5× Native Gel Sample Loading Buffer (NCM biotech) and heated at 100 °C for 10 min. Proteins were separated electrophoretically on 10–15% polyacrylamide gel (Bio-Rad) and transferred to PVDF membrane Trans-Blot Turbo transfer system (Bio-Rad). Before incubating with primary antibodies, the membranes were blocked with PBS containing 5% non-fat milk powder and 0.1% Tween. Then, the membranes were incubated with primary antibodies against IFI35 (1:1000, Abcam, #233415; 1:100, Santa Cruz, #393513), β-Actin (1:5000, CST, #3700S) overnight at 4 °C. After washing with PBST, the membranes were incubated with appropriate HRP-linked secondary antibodies (Anti-Rabbit and Anti-Mouse; Abcam) for 1 h at room temperature. Membranes were washed again and incubated with Pierce ECL Western Blotting Substrate (ThermoFisher) and imaged using ChemiDoc^TM XRS+ System (Bio-Rad).

HepG2.2.15 or HepAD38 cells cultured in a 12-well plate were lysed with 200 μl Laemmli sample buffer (Bio-Rad, PA, USA) supplemented with 2.5% 2-mercaptoethanol (Sigma-Aldrich, MO, USA). Cell lysates were subjected to denaturing gel electrophoresis with 12% Criterion TGX stain-free precast gels and Tris-glycine-SDS running buffer (Bio-Rad). Proteins were transferred from the gel onto a polyvinylidene difluoride (PVDF) membrane Trans-Blot Turbo transfer system (Bio-Rad). Membranes were blocked with 5% nonfat milk in Tris-buffered saline (TBS)-0.1% Tween for 1 h and incubated with the primary antibody overnight at 4°C. After washing with TBS containing 0.1% Tween 20 (TBST), the membrane was incubated with the secondary antibody. Membranes were again washed 3 times with TBST and soaked with 200 μl Clarity Western ECL substrate (Bio-Rad) and imaged with the iBright imaging systems (Thermo Fisher Scientific). The primary antibodies used in the present study include anti-HBc antibody (catalog no. B0586; Dako, United Kingdom), anti-PAPD5 antibody (catalog no. HPA042968; Atlas Antibodies, Bromma, Sweden), and anti-β-actin antibody (catalog no. ab8227; Abcam, Cambridge, United Kingdom).