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).