Enhanced chemiluminescence hrp substrate

Total proteins were extracted from the HepG2 cells treated with OA and/or antioxidant using the M-PER Mammalian Protein Extraction Reagent (Thermofisher, Waltham, MA, USA) and quantified with a Nanodrop spectrophotometer (Nanodrop Technologies, Oxfordshire, UK). Proteins were separated using sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis at 100 V for 110 min; thereafter, they were transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA) using the Mini Trans-Blot^® Electrophoretic Transfer Cell (Bio-Rad Laboratories) at 80 V for 100 min. The membranes were then incubated with TBS-T with 5% skim milk (Becton Dickinson, Sparks, MD, USA), and then incubated with primary antibody which is either one of rabbit Phospho-AMPK alpha-1-, AMPK alpha-1-polyclonal antibody (Invitrogen, Waltham, MA, USA), or mouse anti-beta-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). As secondary antibodies, chicken anti-rabbit IgG-HRP (Santa Cruz Biotechnology) and goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) were used. Signals were detected by exposing the membrane to enhanced chemiluminescence HRP substrate (Thermofisher Scientific) using a Fuji LAS1000 Lumino Image Analyzer (Fujifilm Corporation, Tokyo, Japan) and calculated using an image calculator, ImageJ program.

MNT1 cells (1 × 10⁵ cells/well) in 6-well plates were cultured in complete DMEM with or without 50 μM cajanin for 24–72 h. The cells were harvested and lysed with RIPA buffer containing 1% protease inhibitor and 1% phosphatase inhibitor for 45 min on ice. Then, the equal protein samples determined by BCA assay kit were mixed with loading buffer, heated at 95 °C for 5 min and further subjected to 10% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). The separated proteins in polyacrylamide gels were transferred onto nitrocellulose membranes and blocked with 5% skim milk in TBST (25 mmol/L Tris-HCl; pH 7.4, 125 mmol/L NaCl, 0.1% Tween 20) at room temperature for 1 h. The membranes were incubated overnight with specific primary antibody at 4 °C then washed 3 times × 7 min with TBST. Horseradish peroxidase (HRP)-conjugated secondary antibody was added onto the membrane to interact with respective primary antibody for 2 h at room temperature. The signal from the target protein was visualized by using an enhanced chemiluminescence HRP substrate (Thermo Scientific, Rockford, IL, USA). The intensity of protein signal was quantified using analyst/PC densitometric software (Bio-Rad, Hercules, CA, USA).