Ecl plus western

Immunoblotting was performed as described previously with slight modifications (Kawasaki et al., 1999 (link)). In brief, tissue lysates were separated by SDS-PAGE and transferred onto a PVDF membrane. After blocking with 5% skim milk, the membrane was then incubated overnight with primary antibodies, which included anti-Shh (Cell Signaling Technology, RRID:AB_2188191) and anti-ß-actin (Sigma-Aldrich, RRID:AB_476744) antibodies, and subsequently with horseradish peroxidase-conjugated secondary antibodies. Signals were detected by the ECL Plus Western blotting detection system (GE Healthcare Life Science). Signal intensities of bands were measured using Multi Gauge software (Fujifilm).

The human hepatoma-derived cell line Huh7.5 were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin and 2 mM L-glutamine (Lonza).
J6/JFH1 cDNA (kindly provided by C. Rice, Rockefeller University) was used to generate the HCV cell model. Antibodies anti human KLF15, KLF3, TCF7L2 (Abcam), Adipsin, Adipogenin, Twist 1, beta Actin (Santa Cruz Biotechnology, Santa Cruz, CA), HCV NS5a (Austral Biologicals) were used for immunofluorescence assay. Detection was achieved using horseradish peroxidase–conjugate secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). ECL Plus Western and ECL-Hyperfilm (GE Healthcare Life Sciences) were used. Specific neutral lipids were stained with BODIPY (Invitrogen).