Anti acc

Western blot analysis was conducted to investigate the expression of proteins and their phosphorylation level in hepatic tissues and cells. Hepatic tissues were ground into powders with liquid nitrogen and hepatic cells were homogenized in RIPA lysis buffer (1% leagene PMSF, 1% protease and Phosphatase Inhibitor Cocktail). Proteins extracted from each group were separated by SDS-PAGE and transferred to PVDF membranes. Nonspecific binding was blocked using a blocking reagent (0.1% Tween 20 and 5% Bovine Serum Albumin in TBS) and the membranes were then incubated with specific primary antibodies overnight at 4°C. Primary antibodies (1:1000) used in this manuscript are: anti-AMPK alpha 1 and anti-p-AMPK alpha 2 (S345) obtained from Biohua (Hangzhou, China); anti-GAPDH purchased from Santa Cruz (Biotechnology, Santa Cruz, CA, United States); anti-PPARα obtained from Proteintech (Chicago, CA, United States), anti-ACC was obtained from Abcam (Cambridge, England); anti-GLP-1R (Beijing, China, Bioss), and anti-p-ACC purchased from Affinity Biosciences (OH, United States). After incubating the membrane with the appropriate secondary antibodies for 1 h at room temperature, Perkinelmer western lightning Plus-ECL was used to detect the specific bands. The immunoblots were quantified by densitometric analysis using Quantity One software (Bio-Rad, Hercules, United States).

The cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. The BCA Protein Assay Kit (Pierce, Waltham, MA, USA) was used to determine protein concentrations. SDS-PAGE was used to separate equal quantities of protein, which was then transferred to PVDF membranes (Millipore, Burlington, MA, USA). Membranes were blocked with 5% nonfat milk and incubated with primary antibodies overnight at 4 °C, anti-APOE4 (Abcam, Cambridge, UK; catalog #ab279714,1:1000), anti-ABCA1 (Abcam, Cambridge, UK; catalog #ab18180,1:1000), anti-FAS (Abcam, Cambridge, UK; catalog #ab133619,1:1000), anti-ACC (Abcam, Cambridge, UK; catalog #ab109368,1:1000), anti-SCD1 (Abcam, Cambridge, UK; catalog #ab236868,1:1000), anti-GPT-1 (Abcam, Cambridge, UK; catalog #ab214179,1:1000), anti-SREBP1 (Abcam, Cambridge, UK; catalog #ab313881,1:500), and anti-PPARγ (Abcam, Cambridge, UK; catalog #ab310323,1:1000). Protein bands were detected using an enhanced chemiluminescence (ECL) kit (Thermo Scientific, Waltham, MA, USA) after incubation with suitable horseradish peroxidase-conjugated secondary antibodies.

The total proteins of liver tissues (four rats randomly selected from each group) and cells were extracted from BMMs using RIPA lysis buffer and then quantitated with a Pierce BCA Protein Assay Kit (Thermo Fisher, Waltham, MA, U.S.A.). The proteins were separated by 10% SDS/PAGE and then transferred on to a nitrocellulose membrane, which was then blocked with 5% skim milk. The membrane was then incubated with primary antibodies that included anti-AMPK, anti-p-AMPK, anti-ACC, anti-p-ACC, anti-PPARγ, and anti-SREPBP-2 (Abcam, U.S.A.) overnight at 4°C, and then incubated with a HRP–conjugated secondary antibody (Bioworld, China) for 1 h at room temperature. The immunostained proteins were visualized with ECL-Plus reagent (Millipore, Billerica, MA, U.S.A.).