Extraction of protein
Segments (100 mg) of frozen livers were ground up at 0°C, and then 500 μl diluted cell signaling lysis buffer supplemented with 5 μl protease inhibitor cocktail set III (Millipore Corporation, USA) was added. The mixture was vortexed for 3 cycles of 20 min on/3 min off. The suspended liver tissues were sheared with an ultrasonic cell disruptor and centrifuged at 4°C (14000 rpm for 20 min). The supernatant was stored at -80°C until measurement of the levels of cytokines and phosphorylation of the key protein in the signal pathways by ELISA or MILLIPLEX®MAP assay.