Monoclonal antibody against β actin

The liver tissues were homogenized in protein extraction solution. Lysates were cleared by centrifugation at 12,000 x g for 10 mins at 4°C (two times). Proteins were quantified using the Bradford protein microassay (Bio-Rad, Hercules, USA). Equal amounts of protein from each sample were separated on 10% sodium dodecylsulphate polyacrylamide gels by electrophoresis. The proteins were transferred to polyvinylidene fluoride membranes, blocked in 5% fat-free milk solution for 1 hr, and rinsed 3 times in Tris buffer containing 1% Tween^® 20. The membranes were incubated with 1:1000 dilution of primary antibodies against MAP2K4, ERK, JNK, c-Jun, phospho-MAP2K4 (p-MAP2K4), phospho-ERK (p-ERK), phospho-JNK (p-JNK), and phospho-c-Jun (p-c-Jun) (Cell Signaling Technology, MA, USA) overnight at 4°C. Bound antibody was detected with a horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 hr (Cell Signaling Technology, MA, USA), and immunodetected proteins were visualized using the enhanced chemiluminescence (ECL) system. To confirm the equal amount of protein loaded, the membranes were stripped and reprobed with a monoclonal antibody against β-actin (Protein Tech Group, Chicago, IL, USA). Gel bands were quantitated by densitometry and normalised to β-actin.