2.6. Western Blot Analysis

Western blotting was performed as standard protocol. Protein was extracted from frozen liver samples. A total of 80 ng protein was loaded onto 6%, 10%, and 12.5% SDS-polyacrylamide gels, and the separated proteins were transferred to PVDF membranes. The membranes were incubated overnight at 4°C with primary antibodies followed by incubation with a secondary antibody (1 : 2,000). Finally, the blots were scanned using an Odyssey two-colour infrared laser imaging system (Li-Cor, Lincoln, NE, USA). Western blots were performed using the following antibodies; PPARγ (Cell Signaling Technology), mTOR (Cell Signaling Technology), p-mTOR (Cell Signaling Technology, Ser2448), AMPKɑ (Cell Signaling Technology), p-AMPKɑ (Cell Signaling Technology, Thr172), TNF-ɑ (Cell Signaling Technology), IL-1β (Cell Signaling Technology), Bax (Proteintech), cleaved caspase-9 (Proteintech), Beclin1 (Proteintech), LC3 (Proteintech), and β-actin (Abcam).