*4.14. Western Blotting Analyses*

The hepatic tissues were lysed with RIPA lysis buffer (Servicebio technology, Wuhan, China), supplemented with protease inhibitor (Servicebio), and homogenized with an ultrasonic processor. According to the manufacturer's instructions, the total protein of the liver tissue was extracted with a commercial kit (Servicebio technology, Wuhan, China). Then, the concentration of the protein was measured with a BCA kit (Beyotime technology, Shanghai, China). Then, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane, followed by blocking with 5% skim milk (0.5% TBST) and sealed for 1 h. Additionally, then, PDVF membranes were incubated with primary antibodies against NF-κB (1:1000), p-ERK (1:1000), PI3K (1:1000), p-AKT (1:1000), Caspase-3 (1:1000), Bcl-2 (1:1000), Bax (1:1000), and GAPDH (1:3000) were incubated overnight at 4 ◦C. They were washed with TBST at room temperature on a decolorizing shaker three times. After washing, PVDF membranes were incubated with secondary antibodies (1:3000) at room temperature for 2 h. The antibodies were purchased from Proteintech Group, USA. Finally, they were developed and fixed with developing and fixing reagents, and the Alpha software processing system analyzes the optical density values of the target band.
