Anti ck19

Following culture in MRC-5-CM for 14 days, whole liver cancer cells were lysed on ice in a lysis buffer (RIPA, Beyotime, Shanghai, China) with a protease inhibitor mixture cocktail (Roche, Switzerland). After centrifugation at 12000rpm for 30 minutes at 4℃, the protein concentrations of supernatants in samples were measured by the BCA protein assay (Thermo scientific, Rockford, IL, USA). Equal amounts of protein (50μg) were separated by 10%-12% NUPAGE Bis-tris Gel (Invitrogen, CA, USA) electrophoresis (constant voltage: 120mv) and transferred onto polyvinylidene fluoride (PVDF, 0.45μm) membranes (constant current: 350mA for 70/120 min). After being blocked by Tris-buffered saline and Tween 20 (TBST) buffer containing 5% non-fat powder milk for 2h, the membranes were incubated with primary antibodies overnight on ice. After washing the membranes several times in TBST while agitating, detection was performed using the appropriate secondary HRP-conjugated anti-mouse or anti-rabbit antibody. Immunoreactive bands on the blots were visualized with enhanced chemiluminescence reagent ECL kit (Beit Haemek, Israel). Anti-GAPDH, anti-WNT-2, anti-WNT-5B, anti-WNT16, anti-TGFB1, anti-CTNNB1, anti-IL6, anti-Nanog, anti-OCT4 and anti-CK19 primary antibodies were purchased from (Epitomics).

The WB procedure was performed as described previously
[39 (link)] with some modifications. Briefly, after transient transfection with plasmids, cells were harvested and lysed in a lysis buffer containing a cocktail of protease inhibitors. After centrifugation at 12,000 rpm for 15 min at 4°C, supernatants were collected, mixed with dithiothreitol, and used for WB. The ProteoExtract® Subcellular Proteome Extraction Kit (Millipore) was used for extraction of subcellular fractions following the manufacturer’s protocol. Equal amounts of protein extract were electrophoresed in 10% SDS-PAGE gels and then transferred to nitrocellulose membranes. The membranes were blocked with 5% bovine serum albumin (BSA) at room temperature (RT) for 1 h, incubated with the primary antibody overnight at 4°C, and incubated with the secondary antibody at room temperature for 1 h. Blots were washed in Tris-buffered saline with 0.1% Tween-20 and proteins were visualized by chemiluminescence. The following antibodies were used for WB: anti-SOX1 (IB 1:2000, Epitomics, CA, USA), anti-CK19 (1:8000, Epitomics), anti-CK18 (1:4000, Epitomics), anti-CK13 (1:4000, Epitomics), anti-CK8 (1:4000, Epitomics), anti-Involucrin (1:4000, Abcam, MA, USA), anti-GAPDH (1:10000, ProteinTec Group, IL, USA), anti-c-Myc (N-262) (1:2000, Santa Cruz Biotechnology, CA, USA), and anti-β-catenin (1:2000, Upstate, NY, USA).