Immunoblotting Analysis of HepG2 Cells

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 peroxidase-conjugated 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 (link)].