Western blotting
Human HCC tumor and peri-tumor tissues were lysed in RIPA lysis buffer (Solarbio, Beijing, China) for 30 min and total protein was harvested by centrifugation (12,000 rpm for 10 min) at 4 °C. A total of 25 ug of collected protein were separated by SDS-PAGE and transferred to PVDF membranes. After the membranes with blotted proteins were incubated with rabbit anti-human EBI3 antibody (1:250) and rabbit anti-human IL12A antibody (1:1000) overnight at 4 °C , a horse radish peroxidase)-conjugated goat anti-rabbit IgG antibody (1:6000; Cell Signaling Technology, Danvers, MA, USA) for 30 min at 37 °C. The targeted proteins were detected and quantified using a Chemi Scope 6300 Touch chemiluminescence imaging system (Clinx, Shanghai, China). The membranes were stripped and reblotted with HSP90 antibody to verify the equal loading of protein in each lane. Signals of target or reference protein bands were quantified using Image J software (developed by NIH, MD, USA).