Anti scd1

Protein was extracted from inguinal fat using radio immunoprecipitation assay (RIPA) buffer. The protein concentration was evaluated using the BCA protein assay (ThermoFisher). Briefly, 20 µg total protein was separated on a 10% SDS-PAGE gel. After electrophoresis, total proteins were transferred onto a PVDF membrane, and the PVDF membranes were cut according to the molecular weight of the target protein. The PVDF membranes were blocked with 5% skimmed milk and incubated overnight at 4 °C with primary antibodies against anti-SCD-1, PPARγ (Cell Signaling Technology, Inc.), and C/EBPα (Santa Cruz Biotechnology, Inc). β-Actin protein expression was evaluated as a normalization control (Abcam, Cambridge, UK). After incubation with primary antibodies, anti-mouse IgG secondary antibodies (Cell Signaling Technology) were added to the membranes and incubated for 60 min at room temperature. The blot was scanned in a Fluor ChemM instrument (ProteinSimple, Santa Clara, California, USA). The data were analyzed by Image J software and expressed as fold-change relative to the control group after normalizing against β-actin.

Dulbecco Modified Eagle Medium (DMEM) was obtained from GIBCO (USA). Fetal bovine serum (FBS) was obtained from Biolnd (Aus). The total RNA extraction kit (TRIzol) was purchased from TaKaRa (Jap). Trans Script assay kit was obtained from Beijing Trans Gen Biotech Co. Ltd. (CN) and qPCR assay kit was from Invitrogen (USA). ICAM-1, VCAM-1, IL-6 gene primers were obtained from Life Technologies (USA). Anti-ICAM-1, anti-p38, anti-p-p38, anti-SAPK/JNK, anti-p-SAPK/JNK, anti-ERK 1/2, anti-p-ERK1/2, anti-TLR4 and anti-SCD-1 were purchased from Cell Signaling Technology (USA). TAK242 was purchased from Med Chem Express (USA). Recombinant human leptin was purchased from Peprotech (USA). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphe-nyltetrazolium (MTT), elaidic acid (9t18:1) and vaccenic acid (11t18:1) were purchased from Sigma (St. Louis, USA) with purity over 99%. The CLA isomer (9-cis, 11-trans-CLA) was purchased from Cayman Chemical Company (AnnArbor, MI, USA) with purity over 96%. 9t18:1, 11t18:1 and 9c11t-CLA were dissolved in 0.1 mM sodium hydroxide solution at 65 °C.

Protein extraction and western blot analysis were performed as previously described (Serrano et al., 2012 (link); Vida et al., 2013 (link)). The samples (50 µg of total proteins each) were resolved on 4-15% Ready Gel Precast Gels (Bio-Rad Laboratories, Inc.) and subsequently blotted onto nitrocellulose membranes (Bio-Rad). Specific proteins were detected after incubation in TBS-T containing 2% BSA and the corresponding primary antibodies: rabbit anti-ACCα/β, anti-FAS, anti-SCD1, anti-AMPKα, anti-phospho-AMPKα (Thr172), anti-STAT3, anti-phospho-STAT3 (Tyr705) and anti-actin antibodies (Cell Signaling Technology Inc., MA, USA). Rabbit anti-CPT1a and anti-adaptin γ antibodies were purchased from Abcam (Cambridge, UK). An anti-rabbit HRP-conjugated antibody was used as secondary antibody (Promega, Madison, WI, USA). The specific protein bands were revealed using the enhanced chemiluminescence detection system (Santa Cruz, Biotechnology Inc., CA, USA), according to the manufacturer's instructions, and the images were visualized using an Autochemi-UVP Bioimaging System. The bands were quantified through densitometric analysis using ImageJ software (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, MD, USA, http://imagej.nih.gov/ij, 1997-2012). The levels of specific proteins were normalized to actin or adaptin levels.

For immunoprecipitation experiments, cells were lysed in buffer (100 mM Tris-HCl pH 8, 100 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM DTT, protease inhibitors (Complete, Roche)). 1 mg of total cellular protein was incubated overnight at 4 °C with 1 μg of anti-p53 antibody (1C12, Cell Signalling) and protein G-Dynabeads (Life sciences). IP extracts were analyzed by immunoblotting with anti-p53 (1C12, Cell Signaling, 1/1000) and anti-E4F1 (1/2000) antibodies^{21 (link)}. For total protein extracts, cells were lysed in Laemmli buffer (80 mM Tris pH=6,8, 2% SDS, 12% sucrose, 2% β-mercaptoethanol, bromophenol blue) and immunoblotting was performed using the following antibodies: anti- SCD1 (Cell Signaling, 1/1000), FASN (Santa Cruz, 1/1000), ACC (Cell Signaling, 1/1000), Catalase (Santa Cruz, 1/1000), PPARγ (Santa Cruz, 1/500), C/EBPα (Santa Cruz, 1/1000), aP2 (Santa Cruz, 1/1000), γH2AX (Millipore, 1/1000) and ACTIN (Sigma, 1/7000). Oxyblots were carried out according to the manufacturer’s instructions (Millipore). Adobe Photoshop CS4 was used to crop images from unprocessed images. Uncropped images are provided in the Source Data file.

Protein isolation was performed with RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycolate, 1 mM EDTA pH 8 and 1 tablet of protease inhibitor cocktail Roche Complete (Roche) and 1 tablet of phosphatase inhibitor PhosSTOP (Roche) added per 10 ml of buffer. In total, 40 μg of protein was resolved under denaturing conditions (SDS-PAGE) and transferred to the PVDF membrane, and the membrane was blocked in Odyssey blocking buffer (Li-Cor Bioscience). Blots were probed with anti-HDAC3 (cat no 2632, Cell Signalling Denver, CO, 1:1000 or Abcam, Cambridge UK, AB93172, 1:1000), anti-HSD17B4 (Novus Biologicals, Colorado, US, NPB1-33192, 1:2500), anti-SCD1 (Cell Signaling, CST2784s, 1:2500) at 4 °C overnight or anti-β-actin (cat no A5316, Sigma-Aldrich, St. Louis, MO, 1: 10,000) at room temperature 1 h. Secondary antibodies used were IRDye^® 800CW Goat anti-Mouse IgG (P/N: 926-32210, 1:10,000) and IRDye^® 680RD goat anti-rabbit IgG (P/N: 926-68071, 1:10,000, Li-Cor Bioscience, Nebraska, USA). The infrared fluorescence image was obtained using Odyssey infrared imaging system (Li-Cor Bioscience). Uncropped blots are available in the source data file.

Formalin-fixed, paraffin-embedded tissue was cut into 4 μm sections and analyzed by IHC, as described previously [27 (link)]. In brief, anti-SCD1 (diluted 1:100, Cell Signaling Technology, Danvers, MA, USA, catalog number: #2794), anti-β-catenin (diluted 1:100, Abclonal, Woburn, MA, USA, catalog number: A19657), anti-CYP19A1 (diluted 1:100, Abclonal, catalog number: A2161), and anti-Ki67 (diluted 1:500, Servicebio, catalog number: GB111141) were used as primary antibodies to incubate the tissue sections after heat-induced epitope retrieval (in 10 mM sodium citrate buffer of pH 9.0), then incubated with secondary antibody (diluted 1:100, G1213, Servicebio), followed by diaminobenzidine. The slides were counterstained with hematoxylin. The researchers scored the slides for the expression level of SCD1, β-catenin, and CYP19A1 in a double-blind manner, and the scoring system was reasonably adjusted according to the previous description [27 (link)].