Western blot analysis

HepG2 cells were seeded at 1 × 10⁶ cells/mL in a 6-well culture plate. After 24 h for incubation, the cells were washed with the serum free medium (free phenol red) and treated with the BF of Cur or CurDD for 24 h. The treated cells in the 6-well plate were resuspended in an ice-cold lysis buffer for 30 min at 4 °C and centrifuged at 13,500 g at 4 °C for 5 min. Equal amounts (40 µg) of each protein sample were applied to 10% sodium dodecyl sulfate polyacrylamide gels and subjected to electrophoresis. Then, the protein samples were transferred to a pure nitrocellulose membrane (Amersham™Protran®, Sigma Aldrich), and blocked with 5% dry milk. The membrane was incubated with primary antibodies against cleaved caspase-3 (1:1000), cleaved caspase-9 (1:1000) Bax (1:1000), Bcl-2 (1:1000), LC3B (1:1000) or β-actin (1:5000) at 4 °C overnight. Then, membranes were washed with Tris buffered saline with Tween 20 and incubated with species-specific horseradish peroxidase conjugated secondary antibody reacted with Super Signal solution (Endogen Inc, Rockford, IL, USA) for 2 min. The membrane was exposed to an X-ray film, stripped off the bound antibody and re-probed with anti-β actin antibody to confirm the equal loading of protein. The density of target bands was quantified with the Image J program (downloaded from http://rsb.info.nih.gov/ij/). Results were expressed as a relative ratio of band intensity of the target proteins and β-actin.