Bca protein assay kit

Cells were inoculated into 96-well plates. The original cell culture medium was discarded after each group of cells was treated, followed by rinsing three times with DPBS. Next, 100 µL of cell lysis solution was added to each well and oscillated to fully lyse the cells. Intracellular TG level was measured with GPO-POD method using Triglyceride Colorimetric Assay Kit (Nanjing, Jiangsu, China). The content of protein in each sample was determined by BCA Protein Assay Kit (Nanjing, Jiangsu, China).

The liver tissue samples were homogenized in RIPA buffer with 1% of protease inhibitor cocktail and centrifuged at 14000 g for 10 min at 4°C. Then, the supernatant was removed and obtained total proteins. Besides, the protein of cytoplasmic and nucleus was extracted by the extraction kit (Thermo). And to determine the protein concentrations, the BCA Protein Assay Kit (Nanjing, Jiancheng) was used. The equivalent proteins of liver samples were separated by SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane. After that, the membranes with 5% skimmed milk were blocked under the room temperature for 1 h and then incubated with primary antibody of CYP2E1 (1 : 2000), Nrf2 (1 : 1000), HO-1 (1 : 1000), NQO1 (1 : 1000), TLR4 (1 : 3000), NF-κB P65 (1 : 1000), and β-actin (1 : 3000) at 4°C overnight, respectively. Next, each membrane with an HRP-conjugated secondary antibody was incubated under the room temperature for 2 h. Subsequently, the membranes with ECL reagent were visualized and detected by Western Blotting Detection System. The density of each band was analyzed using ImageJ software.