Mouse anti afp

L-02 and HLE cells were transfected with pcDNA3.1-HBx for 24 h and 48 h, and L-02-X and HLE-X cells were transfected with AFP-siRNA vectors for 48h, the expression changes of AFP, Ras and CXCR4 in L-02 and HLE cells were evaluated by Western Blotting analysis. Briefly, total proteins were extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China). The proteins (50μg total) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After incubating with 5% skim milk in Tris-buffered saline and Tween-20(TBST) at 37°C for 30 min, the membranes were probed for the following primary antibodies: mouse anti-AFP(1:500), Ras(1:500) or β-actin(1:1000); rabbit ant-CXCR4(1:500) antibody(all from Santa Cruz Biotechnology Inc.) overnight at 4°C. After three washes with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at 37°C. The bands were visualized using enhanced chemiluminescence reagents (Thermo Fisher, Rockford, IL, USA) and analyzed with a gel analysis system (VersDoc TM5000MP System; BIO-RAD, Guangzhou, China). The expression of β-actin was used as loading control.