Anti fas

The protein expression in the liver was determined through Western blotting following a previously described protocol [20 (link)]. The membranes were then incubated with primary antibody (PPARγ, IL-6, IL-1β, SIRT1, LXRα, pAMPKα, AMPKα, PPARα, pACC, ACC, FAS, SREBP1c, CPT1B, and β-actin) at room temperature for 2 h. In this study, the primary antibodies were anti-PPARγ (Cat# 2435S, 1:1000, Cell Signaling, Danvers, MA, USA), anti-IL-6 (Cat# 21865-1-AP, 1:1000, Proteintech, Rosemont, IL, USA), anti-IL-1β (Cat# 16806-1-AP, 1:1000, Proteintech), anti-SIRT1 (Cat# 9475S, 1:1000, Cell Signaling), anti-LXRα (Cat# ab176323, 1:1000, Abcam, Cambridge, UK), anti-pAMPKα (Cat# AF3423, 1:1000, Affinity, San Francisco, CA, USA), anti-AMPKα (Cat# AF6423, 1:1000, Affinity), anti-PPARα (Cat# sc-398394, 1:500, Santa Cruz, Santa Cruz, CA, USA), anti-pACC (Cat# D7D11,1:2000, Cell Signaling), anti-ACC (Cat# C83B10, 1:2000, Cell Signaling), anti-FAS (Cat# C20G5, 1:2000, Cell Signaling), anti-SREBP1c (Cat# ab28481, 1:2000, Abcam), anti-CPT1B(Cat# DF3904, 1:500, Affinity), and anti-β-actin (Cat# GTX109639, 1:10000, Genentech, San Francisco, CA, USA). The relative expression of proteins was quantified densitometrically using ImageJ software (Wayne Rasband, Madison, WI, USA), and β-actin was used as the internal control.

The paraffin-embedded tissue sections were deparaffinized with xylene and dehydrated in gradually decreasing concentrations of ethanol. For immunohistochemical analysis, the dehydrated tissue sections were immersed in 10 mM sodium citrate buffer (pH 6.0) for 5 min at 95 °C. The last step was repeated using 10 mM fresh sodium citrate solution. The sections were allowed to cool in the same solution for 20 min and then rinsed with PBS. Next, the sections were incubated with a primary antibody for 1 h at 37 °C. The primary antibodies were anti-PPAR-γ (Cell Signaling Technology, Danvers, MA, USA), anti-SREBP-1 (Abcam, Cambridge, Cambridgeshire, UK), anti-FAS (Cell Signaling Technology, Danver, MA, USA), and anti-ACC (Cell Signaling Technology). After three rounds of serial washing with PBS, the sections were processed with an indirect immunoperoxidase technique using a commercial EnVision System kit (DAKO, Carpinteria, CA, USA). The slides were examined with a Pannoramic^® MIDI slide scanner, and integrated optical density was analyzed using the i-Solution DT software (IMT i-Solution).

Total protein was extracted by RIPA buffer (Solarbio, Beijing, China) containing protease inhibitors (PMSF) (Solarbio, Beijing, China), while nuclear protein was extracted using a Nuclear Protein Extraction kit (Solarbio, Beijing, China). The protein concentrations were determined using a BCA Protein Assay kit (Solarbio, Beijing, China). The tissue lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Pall Co, USA) at a voltage of 90V. Blotted membranes were blocked with 5% BSA for 1.5 h at room temperature and incubated overnight at 4°C with anti-FAS(#3180, Cell Signaling Technology), anti-SREBP-1(#NB100-2215, Novus), anti-NRF2(#12721, Cell Signaling Technology), anti-AHR (#sc-13308, Santa Cruz Biotechnology), anti-Lamin-B2(#12255, Cell Signaling Technology) and anti-β-Actin(#3700, Cell Signaling Technology), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (1:5,000, Zhongshan Golden Bridge Biotechnology, Beijing, China) for 1 h at room temperature. Fluorescent bands were visualized and photographed using an SYNGENE CGQ/D2 GEL-Image System (Bio-Rad, America).

Total proteins were isolated from white adipose tissues using RIPA (Solarbio Life Science, Beijing, China) buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were determined using a protein assay reagent (Beyotime Biotechnology, China) and equal amounts of protein (60 μg) were loaded in each well of an 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel. After the proteins were transferred onto a polyvinylidene difluoride membranes, the blots were blocked with 5% bovine serum albumin, followed by incubation overnight at 4°C with the following primary antibodies: anti-AMPKα (23A3), anti-phospho-AMPKα (Thr172), anti-ACC, anti-FAS, and anti-CPT1α (Cell Signaling Technology, United States). After three washes with TBST buffer, the blots were incubated with appropriately diluted horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The immunoblots were visualized with an enhanced chemiluminescence detection kit (Tiangen Biotech, Beijing, China). Protein levels were normalized to tubulin as a loading control.

FBT was purchased from Shaanxi Jingwei Fuzhuan Brick Tea Co., Ltd. (Shaanxi Jingwei Fuzhuan Brick Tea Co., Ltd., Xianyang, China). DEAE-cellulose DEAE-52 (3 cm × 12 cm, 0.45 μm), cellulase, papain, α-glucosidase (≥700,000 U/mL), acarbos (95%), PNPD, α-amylase (50 U/mg), Sephadex G-200 (Φ20 × 600 mm), and microporous membranes were obtained from Shanghai Yuanye Biotechnology Co., Ltd. (Yuanye Biotechnology Co., Ltd., Shanghai, China). DMEM high-glucose medium, penicillin–streptomycin, and trypsin-EDTA were obtained from Bioengineering Co., Ltd. (Bioengineering Co., Ltd., Shanghai, China). Oil Red O staining solution and blue cell viability dye were obtained from Suolaibao Biotechnology Co., Ltd. (Suolaibao Biotechnology Co., Ltd., Beijing, China). Anti-β-actin, anti-SREBP-lc, anti-FAS, anti-AMPK, and anti-P-AMPK were obtained from Cell Signaling Technology (Cell Signaling Technology, Boston, MA, USA). All other reagents were of analytical grade and were obtained from Tianli Chemical Reagent Co., Ltd. (Tianli Chemical Reagent Co., Ltd., Tianjin, China).