*4.7. Immunoblotting*

After treatment of HepG2 liver cancer cell lines with different concentrations of EO for 24 h, cells were harvested using trypsin and washed twice with ice-cold PBS. For the immunoblotting analysis, treated cells were lysed in RIPA buffer (Santa Cruz Biotech, CA, USA) for 14 min on ice, then centrifuged at 8000× g for 15 min at 4 ◦C. Supernatants

were collected and estimated using Bradford reagent. An equal amount of protein was denatured at 92 ◦C for 7 min. Denatured proteins were then separated on SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk at room temperature for 30 min, incubated with primary antibodies (BCL-2, 1:1000; CASPASE-3, 1:2000; CYP-1A1, 1:1000; and NFκB, 1:1500) from cell-signaling technology (CST; Beverly, MA, USA) overnight at 4 ◦C, and then washed and incubated with horseradish peroxidaseconjugated secondary antibody at room temperature for 1 h. Protein bands were visualized by enhanced chemiluminescence (Hisense, Thermo Scientific, Waltham, MA, USA) and analyzed using a Licor analyzer. β-actin (1:2000) was used as an internal control [54].
