Srebp 1c

Antibodies for Akt, phospho-Akt (Ser⁴⁷³), AMP-activated protein kinase (AMPKα), phospho-AMPKα (Thr¹⁷²), the Akt substrate regulating GLUT4 translocation (AS160), phospho-AS160 (Thr⁶⁴²), ERK1/2, phospho-ERK1/2 (Thr²⁰²/Tyr²⁰⁴), p38 mitogen-activated protein kinase (p38), phospho-p38 (Thr¹⁸⁰/Tyr¹⁸²), IRS-1, IL-6, MCP-1, Na⁺/K⁺-ATPase, TNF-α, and α-Tubulin were purchased from Cell Signaling (Danvers, MA, USA). Ang II, IL-17A, COX-2, c-Jun-N-terminal kinase (JNK), phospho-JNK (Thr¹⁸³/Tyr¹⁸⁵), mineralocorticoid receptor (MCR), PPARɣ, C/EBPα, adipocyte protein (aP2), adiponectin, β-actin, α-adducin-1(ADD1), cytochrome P450 family 11-subfamily β-2 (CYP11β-2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz (Dallas, Texas, USA). Antibodies for leptin, mTOR, and SREBP-1c were obtained from Abcam (Cambridge, MA, USA). For rt-PCR, the primers of target cDNAs such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) were purchased. (GenoTech Corp, Dajeon, Korea) ELISA kits for detecting the cytokines such as MCP-1, IL-6, and TNF-α were purchased from BioLegend Inc (San Diego, CA, USA). U0126 or PD98059, f MEK/ERK inhibitors were purchased from Sigma Aldrich (St. Louis, MO, USA)

Western blots were performed as described previously [20 (link)] for AMP-activated protein kinase (AMPK; Cell Signaling Technology, Danvers, MA, USA), p-AMPK (Cell Signaling Technology), cleaved caspase 3 (Cell Signaling Technology), sterol regulatory element-binding proteins 1c (SREBP1c; Abcam, Cambridge, UK), β-actin (Abcam), and fatty acid synthase (FAS; Santa Cruz Biotechnology, CA, USA).

Protein was extracted from HK-2 PTCs lysate and western blotting were performed as previously described [36 (link)]. Briefly, 15 mg of total cell protein was mixed with 6X Laemmli sample buffer containing mercapto-ethanol and heated at 95 °C for 10 min. Samples were then analyzed by SDS-PAGE in 2.5 to 10 % Novex Bolt mini gels (Life Technologies, California, USA) and electroblotted to Hybond Nitrocellulose membranes (Amersham Pharmacia Biotech, Bucks, UK). Membranes were blocked in Tris-buffered saline containing 0.2 % Tween 20 (TTBS) in 5 % skim milk for 2 h. Then primary antibodies against FXR 1:1000 (Abcam); fibronectin 1:1000 (Sigma Aldrich), collagen IV 1:5000 (Abcam) and SREBP1c 1:1000 (Abcam) were added and incubated overnight at 4 °C. Membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature, and washed. The membranes then were reprobed with β-Actin 1:10000 (Santa Cruz, CA). Protein bands were visualized using the enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech). The bands corresponding to fibronectin (220 kDa), collagen IV (250 kDa), FXR (69 kDa), SREBP1c (120 kDa) and β-Actin (42 kDa) were captured using LAS 4000 (Fujifilm, Tokyo, Japan), corrected for β-Actin as a loading control. They were then analyzed using ImageJ software (v1.26r, National Institutes of Health, USA).

The liver tissues were lysed with RIPA buffer containing protease inhibitors (Beyotime, Shanghai, China) and centrifuged at 4 °C for 15 min at 5000 g. Subsequently, the supernatants were collected. Equal amounts of protein, determined by a BCA protein assay (BCA protein assay kit, Beyotime, Shanghai), were separated using a 10% SDS-polyacrylamide gel. After SDS-PAGE, the proteins were transferred to a polyvinylidene fluoride membrane following the manufacturer’s instructions. The membrane was blocked with 5% (wt/vol) skim milk in Tris-buffered saline (TBS)/Tween 20 for 1 h at room temperature and incubated overnight at 4 °C with FASN antibody and β-actin (Cell Signaling Technology, MA, USA), SREBP-1c, and AMPK/p-AMPK (Abcam, MA, USA). This was followed by incubation with peroxidase-conjugated anti-rabbit IgG antibody (Sigma, St Louis, MO, USA) in a blocking solution according to the manufacturer’s instructions and then visualization with a chemiluminescence reagent (Amersham Bioscience, Piscataway, NJ, USA). Based on an analysis of the gray intensity, the semi-quantitative protein expression was analyzed using AlphaView software.

The protein lysates of ESCC cells were isolated after the cells were treated with shFBP1. The concentration of the protein was quantified and transferred the into nitrocellulose (NC) membranes. The NC membranes were blocked, and incubated with the primary antibody for 2h at room temperature. Finally, the protein expression was visualized using the chemiluminescence detection system. The primary antibodies against FASN (1:1000; R&D Systems), ACC1 (1:1000; Cell Signaling Technology), SREBP1C (1:2000; Abcam), FBP1 (1:1000; Abcam), a secondary antibody (anti-rabbit IgG, 1:7500; Cell Signaling Technology), and β-actin antibody (1:5000; Abcam) were used to determine the indicated protein expression. The Image J (NIH) was used for the quantification of protein expression.

A normal diet was purchased from Jiangsu Synergetic Pharmaceutical Bioengineering Co., Ltd. (Nanjing, Jiangsu, China, XTADM001) and consisted of 4% fat, 73.1% carbohydrate, and 14.2% protein. A high-fat diet was purchased from Jiangsu Synergetic Pharmaceutical Bioengineering Co., Ltd. (Nanjing, Jiangsu, China, XTHF60) and consisted of 60% fat, 20% carbohydrate, and 20% protein. Metformin was purchased from Sigma (St. Louis, MO, USA, D150959-5G). Hematoxylin eosin (H&E) and oil red O (ORO) staining kits were purchased from Solarbio (Beijing, China, G1120 and G1261). The rabbit antimouse adiponectin (ADIPOQ), MCP-1, TNF-α, IL-6, and DAB horseradish peroxidase chromogenic kits were purchased from Beyotime (Shanghai, China). The rabbit antimouse adiponectin receptor II (ADIPOR2), phospho-AMPKα1, AMPKα1, SIRT1, phospho-NF-κB p65, NF-κB p65, SREBP1C, FAS, acetyl coenzyme A carboxylase (ACC), PPAR-γ, SCD1, β-actin, and goat antirabbit IgG antibodies were purchased from Abcam (Cambridge, UK).