Bca assay kit

The protein of liver tissues and HepG2 cells was lysed and extracted using Radio Immunoprecipitation Assay (RIPA) lysis buffer (Beijing Beyotime, China), and the concentration was quantified using a BCA assay kit (Solarbio, PC0020, Beijing, China). The denatured protein samples (50 µg) from each group were loaded on 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto polyvinylidene fluoride membranes (PVDF) (Millipore, Bedford, MA, USA). Each PVDF membrane was blocked with 1×TBST containing 5% skimmed milk and incubated with primary antibodies at 4°C refrigerator overnight and incubated with secondary antibodies for 1 h in the dark. All the primary antibodies in the Western blot experiment are as follows: PGC1α (1:1000, Proteintech), PPARα (1:1000, Proteintech), PINK1 (1:1000, Proteintech), Parkin (1:1000, Affinity), ATG7 (1:1000, Affinity), SQSTM1/p62 (1:1000, Affinity), FAS (1:1000, ab128856), SREBP-1c (1:1000, A15586), LC3 I/II (1:1000, AB Clonal), and β-actin (1:10000, AB Clonal). The chemiluminescence intensity of all membranes was visualized with enhanced chemiluminescence system. The gray value of protein strips was analyzed using the Image J software.

Cells were lysed in Pierce IP Lysis Buffer (#87788, Thermo Scientific) and protease inhibitor cocktail. Then proteins were quantified using BCA Assay Kit (Solarbio, Beijing, China). For immunoprecipitation analysis, 500 μg of protein from cells were prepared. To preclear the sample, the samples were incubated with 20 μL of Protein A&G magnetic beads (Bimake, Shanghai, China) for 1 h at 4 °C with constant rotation. Simultaneously, 2 μg antibody was incubated with 30 μl magnetic beads at 4 °C overnight with constant rotation. The magnetic beads were then washed 4 times with IP buffer, and immunoprecipitated proteins were eluted from magnetic beads by 1x loading buffer (CWBIO, Beijing, China) and then were incubated at 100 °C for 5 min. The immunoprecipitated proteins were subject to Western blot analysis.

Cells were lysed with RIPA lysate (Beyotime, Shanghai, China), and the concentration of protein was determined by the BCA assay kit (Solarbio, Beijing, China). Briefly, the equal amount of protein was separated with 10% SDS-PAGE (Epizyme, Shanghai, China), and transferred to PVDF membranes (Millipore, MA, USA). The PVDF membranes were blocked with 5% skim milk and incubated at 4 °C overnight with primary antibody, including anti-Fibronectin (Cell Signaling Technology, MA, USA), anti-N-cadherin (CST), anti-E-cadherin (CST), anti-MMP2 (CST), anti-MMP9 (CST), anti-Cytokeratin (Proteintech, Wuhan, China), anti-Vimentin (CST), anti-Ran (Proteintech), anti-Akt (CST), anti-p-Akt (CST), GSK3β (CST), anti-p-GSK3β (CST), anti-β-catenin (CST). β-actin (CST) was used as internal controls. Then the PVDF membranes were incubated with a secondary antibody for 1 h. The blots were scanned by Amersham Imager 600 (GE, Boston, MA, USA). Each sample was repeated three times and the three independent western blot bands were quantitatively analyzed by Image J software (NIH, Bethesda, Maryland, USA).

Proteins were isolated by lysing the cells with RIPA buffer (150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, 1% PMSF, and a cocktail inhibitor for proteases and phosphatases), and the concentration of protein was determined by the BCA assay kit (Solarbio, Beijing, China). After SDS-PAGE separation, protein was transferred to a PVDF membrane (Millipore, Burlington, MA, USA). The PVDF membrane was blocked with 0.5% BSA for 2 h, and then incubated with the primary antibody for 2 h at room temperature. After TBST wash, the PVDF membrane was incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h at room temperature. The membrane was visualized by ECL plus (Bio-rad, Hercules, CA, USA) and examined with G:BoxChemiXL1.4 (Syngene, Cambridge, UK).