Tissue protein extraction reagent

Liver tissues were homogenized using a Tissue Protein Extraction Reagent (Beyotime, Shanghai, China) for 30 min at 4°C. The homogenates were centrifuged at 12,000 g for 15 min at 4°C, and the protein concentration was determined using a Bicinchoninic Acid (BCA) Protein Assay Kit (Beyotime, Shanghai, China). Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose (NC) membranes. After blocking with 5% nonfat milk in TBST for 2 h at room temperature, the membranes were incubated overnight with anti-P53, anti-Bcl-2 (Wanlei Biology, Shenyang, China), anti-Bax, and anti-β-actin (Sangon Biotech, Shanghai, China) primary antibodies. The membranes were then washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibody (ImmunoWay, Plano, USA) at room temperature for 2 h. Following another wash with TBST, the blots were developed using a Meilunbio® fg super-sensitive ECL luminescence reagent (Meilunbio, Dalian, China) and imaged using the Tanon 5200 Imaging System (Tanon Science & Technology Co., Shanghai, China). The relative density of the target bands was quantified using ImageJ software.

Cell lysate was prepared by using modified-RIPA buffer (Tris 50mM, NP-40 1%, NaDOC 0.25%, NaCl 150mM, EDTA 1mM, SDS 0.1%, NaF 5mM, NaVO3 0.4 mM, and protease inhibitors). Tissue proteins were extracted with the tissue protein extraction reagent (Beyotime, Shanghai) supplemented with protease inhibitors cocktail. Protein was quantitated by using the BCA protein concentration kit (Beyotime). Samples were separated by using 10% SDS-PAGE. After transferred to a PVDF membrane, immunoblotting was carried out using various specific antibodies and visualized with enhanced chemiluminescence and the Image Reader LAS-4000 (Fuji, Japan).