Anti fasn

Manufactured by Cell Signaling Technology

Sourced in United States, Switzerland

Anti-FASN is a laboratory product that detects the fatty acid synthase (FASN) protein. FASN is an enzyme involved in the synthesis of fatty acids. This product can be used to analyze the expression and localization of FASN in various biological samples.

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Rabbit anti-FASN Anti-Fatty Acid Synthase (FASN) Rabbit anti-FASN (C20G5, Anti-FASN antibody

26 protocols using anti fasn

Immunoblotting Antibody Panel Validation

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The following antibodies were used for immunoblotting. Abcam: anti-PMP70 (1:1,000 dilution; ab3421), anti-FASN (1μg/ml dilution; ab22759); Cell Signaling: anti-cytochrome oxidase IV (1:250 dilution; 4850), anti-catalase (1:800 dilution; 12980), anti-centromere protein A (1:400 dilution; 2186), anti-calreticulin (1:200 dilution; 12238), anti-FASN (1:1,000 dilution; 3180); Santa Cruz Biotechnology: anti-C13orf31 (1:200 dilution; sc-374553; E7 and 1:500 dilution; sc-376231; E12), anti-caspase 1 p20 (1:250 dilution; sc-1218-R); R&D Systems: anti-IL-1β (1:250 dilution; AF-401-NA); Sigma: anti-β-actin (1:10,000 dilution; A5060). All antibodies used have validation profiles on either Antibodypedia or 1DegreeBio.
The following reagents were used: M-CSF (Peprotech, 300-25), LPS (from Escherichia coli K12, InvivoGen, tlrl-peklps), human IFN-γ (Peprotech, 300-02), murine IFN-γ (Peprotech, 315-05), human IL-4 (Peprotech, 200-04), murine IL-4 (Peprotech, 214-14), ATP (Sigma Aldrich, A2383), C75 (Sigma Aldrich, C5490), etomoxir (Sigma Aldrich, E1905), MitoTEMPO (Sigma Aldrich, SML0737), oligomycin (Sigma Aldrich, O4876), FCCP (Sigma Aldrich, C2920), rotenone (Sigma Aldrich, R8875), antimycin A (Sigma Aldrich, A8674), palmitate-BSA (Seahorse Bioscience, 102720-100), zymosan A (Sigma Aldrich, Z4250), PMA (Sigma Aldrich, P1585), HRP (Sigma Aldrich, P8375), luminol (Sigma Aldrich, A8511).

Cader M.Z., Boroviak K., Zhang Q., Assadi G., Kempster S.L., Sewell G.W., Saveljeva S., Ashcroft J.W., Clare S., Mukhopadhyay S., Brown K.P., Tschurtschenthaler M., Raine T., Doe B., Chilvers E.R., Griffin J.L., Kaneider N.C., Floto R.A., D’Amato M., Bradley A., Wakelam M.J., Dougan G, & Kaser A. (2016). C13orf31 (FAMIN) is a central regulator of immunometabolic function. Nature immunology, 17(9), 1046-1056.

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Immunohistochemical Analysis of BPH and PCa

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The collected tissue samples were immersed in 10% paraformaldehyde overnight and then sliced into 5-μm-thick sections. Immunohistochemical staining of BPH and PCa tissues was analyzed using the method we previously reported [13 (link)]. The sections were incubated with anti-Ki-67(Cell Signaling Technology), anti-PLCɛ (Santa Cruz), anti-p-AMPKα (Santa Cruz), anti-SREBP-1 (Abcam), or anti-FASN (Cell Signaling Technology). The data were analyzed using methods we previously reported [13 (link)].

Zheng Y., Jin J., Gao Y., Luo C., Wu X, & Liu J. (2020). Phospholipase Cɛ Regulates Prostate Cancer Lipid Metabolism and Proliferation by Targeting AMP-Activated Protein Kinase (AMPK)/Sterol Regulatory Element-Binding Protein 1 (SREBP-1) Signaling Pathway. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 26, e924328-1-e924328-12.

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Western Blot Analysis of Signaling Proteins

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Primary antibodies used in this study were purchased from Cell Signaling Technology (Beverly, MA): anti-pPKA^Thr198/PKA, anti-pCREB^Ser133/CREB, anti-pSTAT3^Tyr705/STAT3, anti-pSrc^Ser17/Src, anti-pACC ^Ser79/ACC, anti-pAkt^Ser473/Akt, anti-pAMPK ^Thr172/AMPK, anti-p4EBP1^Thr37/46/4EBP1, anti-pmTOR^Ser2448/mTOR, anti-pP70S6K^Thr389/P70S6K, anti-EPAC-1, anti-BEATA2_AR, anti-FAK, anti-FASN, anti-GLUT4, anti-OCT3, anti-NOTCH, anti-PGC1, anti-pPRAS40^Thr246, anti-PRAS40, anti-pRAPTOR^Ser792, anti-RAPTOR, and anti-PI3K110α. Anti-BCL-2 was purchased from BD Bioscience (San Jose, CA). Anti-P27 was purchased from Thermo Fisher Scientific (Waltham, MA). Anti-rabbit immunoglobulin-horseradish peroxidase-conjugated secondary antibody, LumiGLO reagent with peroxide and cAMP assay kit were also purchased from Cell Signaling Technology, Inc. (Beverly, MA). Anti-IGF1Rα, anti-HMGCR, anti-P21, anti-SCD1, and anti-SCEBP1 were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX); mouse anti-β-actin primary antibody was obtained from Sigma Aldrich (St. Louis, MO). The 1-methyl-1-nitrosourea (MNU) was obtained from Ash Stevens (Detroit, MI) and stored at −80°C prior to use. Metformin and buformin were obtained from Waco Pure Chemical Industries, (Waco, TX); phenformin was obtained from Sigma Aldrich (St. Louis, MO).

Zhu Z., Jiang W., Thompson M.D., Echeverria D., McGinley J.N, & Thompson H.J. (2015). Effects of Metformin, Buformin, and Phenformin on the Post Initiation Stage of Chemically-Induced Mammary Carcinogenesis in the Rat. Cancer prevention research (Philadelphia, Pa.), 8(6), 518-527.

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Isolation and Characterization of HsA from H. lyrata

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HsA was isolated from H. lyrata as previously reported (11 (link)). T0901317 was supplied by Cayman Chemical (Ann Arbor, MI, USA). Anti-SREBP1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-FASN, anti-ACC, and horseradish peroxidase-conjugated secondary antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Thiazolyl blue tetrazolium bromide (MTT), GW3965, β-actin antibody, oil red O, hematoxylin, eosin, and other reagents were supplied by Sigma-Aldrich (St. Louis, MO, USA).

Kim J.K., Cho I.J., Kim E.O., Lee D.G., Jung D.H., Ki S.H., Ku S.K, & Kim S.C. (2021). Hemistepsin A inhibits T0901317-induced lipogenesis in the liver. BMB Reports, 54(2), 106-111.

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Immunohistochemical Analysis of Liver Lipogenesis

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Upon euthanasia, mouse liver tissues were collected and fixed in 4% paraformaldehyde (PFA) overnight at 4°C and embedded in paraffin. For antigen retrieval, slides were put into 10 mM sodium citrate buffer (pH 6.0), and placed in a microwave on high for 10 min. Slides were blocked with 5% goat serum and the Avidin-Biotin blocking kit (Vector Laboratories, Burlingame, CA). Primary antibodies were added to the slides and incubated overnight at 4°C. Slides were washed and incubated with a biotin conjugated secondary antibody. Detection was performed with the ABC-Elite peroxidase kit (Vector Laboratories) using the DAB as the substrate. Primary antibodies used in the study include: Anti-FASN and anti-acetyl-CoA carboxylase (anti-ACC) antibodies; both were obtained from Cell Signaling Technology Inc.

Li L., Che L., Wang C., Blecha J.E., Li X., VanBrocklin H.F., Calvisi D.F., Puchowicz M., Chen X, & Seo Y. (2016). [11C]acetate PET Imaging is not Always Associated with Increased Lipogenesis in Hepatocellular Carcinoma in Mice. Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging, 18(3), 360-367.

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Immunoblotting Protocol for Protein Expression Analysis

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For general immunoblotting, cells were lysed in 1% Nonidet P-40 (NP-40) lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 1 mM Na₃VO₄, 1 mM NaF) + complete protease inhibitors (Roche) for 30 min on ice. Lysates were cleared by centrifugation, boiled in 2x sample buffer (Laemmli buffer + 50 μM 2-βME) and separated on 4–20% SDS-PAGE gels (Bio-Rad). Proteins were transferred to nitrocellulose membranes for 10 min using the TransBlot Turbo system (Bio-Rad) and subsequently blocked with Odyssey blocking buffer (LI-COR). Blots were probed with the following Abs: anti-FASN (Cell Signaling Technology #3180), anti-MCL-1 (Cell Signaling Technology #94296), anti-BCL-2 (Cell Signaling Technology #4223), anti-FASL (BD Pharminogen #556372), anti-BIM (Enzo Life Sciences), anti-NUR77 (Biolegend, clone 1E10A15) and anti-β-actin (Sigma-Aldrich). Blots were incubated with IRDye anti-Rabbit or anti-Mouse secondary antibodies (LI-COR) and imaged using the Odyssey CLx instrument. Band intensity quantification was conducted with StudioLite Software (LI-COR).

Voss K., Luthers C.R., Pohida K, & Snow A.L. (2019). Fatty Acid Synthase Contributes to Restimulation-Induced Cell Death of Human CD4 T Cells. Frontiers in Molecular Biosciences, 6, 106.

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Lipid Metabolism Regulation in NF2 Cells

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pBabe-NF2 was obtained from Addgene. Anti-Fasn siRNA (M-040091-01-0005), anti-Acaca siRNA (M-063938-01-0005), anti-Mtor (M-065427-00-0005), anti-Rptor (M-058754-01-0005), anti-Rictor (M-064598-01-0005) and non-silencing (D-001206-13-05) siRNA were purchased from Dharmacon. Individual siRNAs against Mtor (SASI_Mm01-00164496 and -00164492), Rictor (-00137732 and -00137730), Rptor (-00055298 and -00334580), Fasn (-00177858 and -00177854), Acaca (-0011590 and -00115905), and Mlycd (-00028572 and -00028576) were purchased from Sigma-Aldrich. Anti-Merlin antibodies were purchased from Abcam (#ab88957). Lipid synthesis and metabolism antibody kit (includes anti-Fasn, -phospho ACC, -ACC, -Lipin1, -ACLY, -phospho ACLY, -ACSL1, and -ACECS1 antibodies), and anti-Casp3 antibodies were purchased from Cell Signaling Technology. Anti-SREBP1 antibodies were purchased from Santa Cruz Biotechnology. Anti-GAPDH antibodies were purchased from EMD-Millipore.
Cerulenin, C75, luteolin, 5-(tetradecyloxy)-2-furoic acid (TOFA) and 5-iodotubercidin were purchased from Enzo Life Sciences. GSK2194069, dimethylsulfoxyde (DMSO), staurosporin, sodium palmitate, 70% perchloric acid, ammonium formate, acetonitrile, acetyl-coenzyme A lithium salt, malonyl coenzyme A lithium salt, propionyl-coenzyme A lithium salt, and poly-L-lysine were purchased from Sigma-Aldrich.

Stepanova D.S., Semenova G., Kuo Y.M., Andrews A.J., Ammoun S., Hanemann C.O, & Chernoff J. (2017). An essential role for the tumor suppressor Merlin in regulating fatty acid synthesis. Cancer research, 77(18), 5026-5038.

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Molecular Mechanisms of Lipid Metabolism

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The following antibodies and reagents were used: Anti-SREBF1 (Proteintech, 14088-1-AP, 1:1000 for western blotting and 4 μg for ChIP), anti-KLF5 (Santa Cruz Biotechnology, sc-398409X, 1:1000 for western blotting, and 4 μg for ChIP), anti-TP63 (R&D Systems, AF1916-SP, 1:1000), anti-Actin (Santa Cruz Biotechnology, sc-8432, 1:2000), anti-ACLY (Cell Signaling Technology, 4332, 1:1000), Anti-FASN (Cell Signaling Technology, 3180, 1:1000), anti-GAPDH (Cell Signaling Technology, 2118, 1:2000), anti-mTOR (Cell Signaling Technology, 2972S, 1:1000), anti-Phospho-mTOR (S2448) (Cell Signaling Technology, 5536T, 1:1000), anti-Phospho-MEK1 (Ser298) (Cell Signaling Technology, 9128S, 1:1000), anti-MEK1/2(D1A5) Rabbit (Cell Signaling Technology, 9124S, 1:1000), anti-p70 S6 Kinase (Cell Signaling Technology, 9202S, 1:1000), anti-Phospho-p70 S6 Kinase (Cell Signaling Technology, 9205S, 1:1000), anti-mouse IgG-HRP (Jackson ImmunoResearch Laboratories, Inc., 115-035-003, 1:10000), anti-rabbit IgG-HRP (Jackson ImmunoResearch Laboratories, Inc., 111-035-144, 1:10000), anti-goat IgG-HRP (Jackson ImmunoResearch Laboratories, Inc., 705-035-003, 1:10000), HCS LipidTOX™ Green Neutral Lipid Stain (Thermo Scientific, H34475, 1:100), Lipofectamine RNAiMAX (Thermo Scientific, 13778150), and Fatostatin (Cayman Chemical, 12562).

Li L.Y., Yang Q., Jiang Y.Y., Yang W., Jiang Y., Li X., Hazawa M., Zhou B., Huang G.W., Xu X.E., Gery S., Zhang Y., Ding L.W., Ho A.S., Zumsteg Z.S., Wang M.R., Fullwood M.J., Freedland S.J., Meltzer S.J., Xu L.Y., Li E.M., Koeffler H.P, & Lin D.C. (2021). Interplay and cooperation between SREBF1 and master transcription factors regulate lipid metabolism and tumor-promoting pathways in squamous cancer. Nature Communications, 12, 4362.

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Western Blot Analysis of Adipogenesis Markers

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We lysed the cultured cells and homogenized tissues in RIPA buffer (Cat# P0045, Beyotime, China) containing a protease-inhibitor cocktail (Cat# 04693132001, Roche, Canada). After centrifugation at 14,000 rpm for 15 min at 4 °C, the protein concentrations were determined using a BCA protein assay kit (Cat# P0012, Beyotime, China). The cell and tissue lysates were then electrophoretically separated on 10% polyacrylamide gels and transferred onto PVDF membranes. The membranes were subsequently blocked with 5% nonfat dry milk dissolved in Tris-buffered saline/0.1% Tween 20 for 1 h at room temperature. After blocking, membranes were probed with the following diluted primary antibodies: anti-FASN (1:1000; Cell Signaling Technology, USA), anti-NOX4 (1:1000; Abcam, USA), anti-FABP4 (1:1000; Proteintech, China), anti-PPARγ (1:1000; Bioworld, USA), anti-β-actin (1:5000; Bioworld, USA) and anti-GAPDH (1:2000; Abcam, USA). After the membranes were incubated with the primary antibodies at 4 °C overnight, the membranes were incubated with goat anti-rabbit (1:1000, Beyotime, China) HRP-conjugated secondary antibodies and signals were detected using Image Lab software (Bio-Rad, USA).

Hua H., Wu M., Wu T., Ji Y., Jin L., Du Y., Zhang Y., Huang S., Zhang A., Ding G., Liu Q, & Jia Z. (2023). Reduction of NADPH oxidase 4 in adipocytes contributes to the anti-obesity effect of dihydroartemisinin. Heliyon, 9(3), e14028.

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Adipogenesis Regulation by Bilobalide

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Dulbecco’s modified Eagle medium (DMEM), Dulbecco’s phosphate-buffered saline (DPBS), fetal bovine serum (FBS), and 0.25% trypsin were purchased from Gibco (Rockville, MD, USA). Penicillin-streptomycin solution was purchased from Hyclone (Provo, UT, USA). Insulin, dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX), and MTT were obtained from Sigma-Aldrich (St. Louis, MO, USA). 3T3-L1 preadipocytes were purchased from ATCC (Manassas, VA, USA). Bilobalide was obtained from the National Institutes for Food and Drug Control (Beijing, China). Primary antibodies specific for anti-pACC1 (S79), anti-ACC1, anti-FASN, anti-perilipin A, anti-ATGL, anti-C/EBPα, anti-PPARγ, anti-HSL, anti-pHSL (S563), anti-GLUT-4, anti-CPT-1α, anti-AMPK, and anti-pAMPK (T172) were acquired from Cell Signaling Technology (Danvers, MD, USA). Anti-SREBP-1c was acquired from Abcam (Cambridge, UK). Anti-β-Actin and goat anti-mouse and goat anti-rabbit IgG secondary antibodies were obtained from Boster Biological Technology (Pleasanton, CA, USA). The AMPK assay kit was purchased from Cell Signaling Technology.

Bu S., Yuan C.Y., Xue Q., Chen Y, & Cao F. (2019). Bilobalide Suppresses Adipogenesis in 3T3-L1 Adipocytes via the AMPK Signaling Pathway. Molecules, 24(19), 3503.

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