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.