C met

Total liver protein was isolated using RIPA lysis buffer (CWBIO, Beijing, China) containing protease inhibitor cocktail (CWBIO). Equal amounts of protein were separated using 8% SDS‐PAGE gel electrophoresis in a Bio‐Rad Mini‐Protean system. The separated proteins were then transferred to nitrocellulose membrane (Millipore, MA) and incubated with specific primary antibodies (aSMA, ID1, HGF, WNT2, c‐MET, GSK‐3β, Proteintech, Wuhan, China; phosphor‐c‐MET, phosphor‐GSK‐3β, Abclonal, Wuhan, China). After washing and incubation with the secondary antibody, antibody complexes bound to specific liver proteins were visualized using the BIO‐RAD Gel Doc XR+ system (Bio‐Rad, CA).

The expression of proteins in Huh7 and Huh7‐R cells was detected by performing western blot (WB). Total protein from Huh7 cells was extracted applying Total Protein Extraction Kit (Cat: BC3710; Solarbio) as the protocol described. BCA Protein Assay Kit (Cat: PC0020; Solarbio) was used to assess the protein concentration. Protein samples were electrophoresed on 10% SDS/PAGE gels. Then, the separated proteins were transferred onto polyvinylidene fluoride membranes (Cat: ISEQ00010; Merck Millipore, Billerica, MA, USA). Subsequently, the membranes were incubated with the primary antibodies, CXCR4 (1 : 1000, Cat: 11073‐2‐AP; Proteintech, Wuhan, China), Cav‐1 (1 : 1000, Cat: 16447‐1‐AP; Proteintech), p‐Cav‐1 (1 : 2000, Cat: ab75876; Abcam), c‐Met (1 : 1000, Cat: 25869‐1‐AP; Proteintech), p‐c‐Met (1 : 1000, Cat: ab68141; Abcam), Raf‐1 (1 : 1000, Cat: 26863‐1‐AP; Proteintech) and EGFR (1 : 10 000, Cat: 66455‐1‐Ig; Proteintech) at 4 °C for 12 h. After blocking with 5% skim milk, the membranes were incubated with goat anti‐rabbit or goat anti‐mouse horseradish peroxidase‐conjugated secondary antibody (1 : 5000, Cat: SA00001‐2/SA00001‐1; Proteintech). GAPDH antibody (1 : 5000, Cat: 10494‐1‐AP; Proteintech) was used as a reference protein for normalization. The data were analyzed by imagej software (National Institutes of Health, Bethesda, MD, USA).