Anti fas

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Anti-Fas is an antibody that binds to the Fas receptor (also known as CD95 or Apo-1). The Fas receptor is a cell surface protein that plays a key role in the initiation of programmed cell death (apoptosis).

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18 protocols using anti fas

Western Blot Analysis of Metabolic Regulators

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Mouse liver, skeletal muscle or white adipose tissue were dissected and immediately frozen in liquid nitrogen. Whole-cell extracts were prepared using lysis buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Igepal CA-630, 0.2mM Na₃VO₄ and a protease inhibitor cocktail (Roche Diagnostics)). Protein concentration was measured by colorimetric assay (Bio-Rad Laboratories, Richmond, CA, USA) and equal amount of proteins was loaded onto SDS gels. After transfer to polyvinylidene difluoride membranes, proteins were probed with primary antibodies (1 μg/ml), followed by horseradish peroxidase-conjugated secondary antibodies, washed and visualized with SuperSignal West Pico/Dura chemiluminescent substrate (Pierce-Thermo Fisher Scientific, Waltham, MA, USA). Blots were reprobed with β-actin-specific antibody for loading controls.
Anti-IRS-1 (Santa Cruz Biotechnology), anti-IRS-2 (Santa Cruz Biotechnology), anti-SCD-1 (Santa Cruz Biotechnology), anti-IRβ (Santa Cruz Biotechnology), anti-pAkt2 (Cell Signaling Technology, Danvers, MA, USA), anti-SREBP-1 (Santa Cruz Biotechnology), anti-ACCα (Santa Cruz Biotechnology), anti-FAS (Santa Cruz Biotechnology), anti-SREBP-2 (Santa Cruz Biotechnology), anti-HMGCR (Santa Cruz Biotechnology), anti-Albumin (Santa Cruz Biotechnology) and anti-β-actin (Santa Cruz Biotechnology) antibodies were used as primary antibodies.

Huo J., Ma Y., Liu J.J., Ho Y.S., Liu S., Soh L.Y., Chen S., Xu S., Han W., Hong A., Lim S.C, & Lam K.P. (2016). Loss of Fas apoptosis inhibitory molecule leads to spontaneous obesity and hepatosteatosis. Cell Death & Disease, 7(2), e2091-.

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Apoptosis Pathway Regulation by GSK-3β Inhibitor

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GSK-3β inhibitor VIII, N-(4-methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea, was purchased from Calbiochem (San Diego, CA, USA). Cell Counting Kit-8 (CCK-8) was from Dojindo (Tokyo, Japan). Mitochondria isolation kit for cultured cells and RIPA buffer were obtained from Thermo Scientific (Rockford, IL, USA). Annexin V and propidium iodide (PI) for flow cytometry were purchased from BD Pharmingen (San Diego, CA, USA). For the Western blot, the following specific antibodies were used: anti-tau (Invitrogen, Carlsbad, CA, USA), anti-phospho-tau (Ser396) (Invitrogen, Carlsbad, CA, USA), anti-Fas (Santa Cruz Biotech, Delaware, CA, USA), anti-Fas ligand (Santa Cruz Biotech, Delaware, CA, USA), anti-cleaved caspase-8 (Novus Biologicals, Littleton, CO, USA), anti-p38α (Santa Cruz Biotech, Delaware, CA, USA), anti-Daxx (Cell signaling, Beverly, MA, USA), anti-cleaved caspase-3 (Cell Signaling, Beverly, MA, USA), and anti-cytochrome C (Cell Signaling, Beverly, MA, USA).

Kim J.E., Lim J.H., Jeon G.S., Shin J.Y., Ahn S.W., Kim S.H., Lee K.W., Hong Y.H, & Sung J.J. (2017). Extrinsic Apoptosis Pathway Altered by Glycogen Synthase Kinase-3β Inhibitor Influences the Net Drug Effect on NSC-34 Motor Neuron-Like Cell Survival. BioMed Research International, 2017, 4163839.

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Tissue Microarray Analysis of CRC

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Tissue microarray (TMA) blocks covering 21 randomly selected CRC and 6 controls from adjacent morphologically normal tissue were cut into 4-mm sections and arrayed according to the Microarray technique with a semi-automatic ATA-100 Chemicon system (Chemicon’s Advanced Tissue Array).^{18 (link)} TMAs were processed for IHC as previously described,^{11 (link)} using as primary antibodies anti-YY1 (1/500), anti-Fas (1/250) or normal IgG serum (negative control) (all from Santa Cruz Biotechnology, Santa Cruz, CA). Immunostaining was digitized and analyzed by an Aperio Scanscope CS (Aperio, Vista, CA), as previously described.^{11 (link)} The sample collection and use was approved by the Ethics Committee of the Children Hospital of Mexico ‘Federico Gomez’ (Mexico City, Mexico). Both CRC and normal samples were collected by the Department of Pathology of Hospital General Regional No. 25, IMSS and Speciality Hospital CMN ‘La Raza’, IMSS (Mexico City, Mexico). All CRC cases were classified according to the Dukes or TNM staging.

Pothoulakis C., Torre-Rojas M., Duran-Padilla M.A., Gevorkian J., Zoras O., Chrysos E., Chalkiadakis G, & Baritaki S. (2017). CRHR2/Ucn2 signaling is a novel regulator of miR-7/YY1/Fas circuitry contributing to reversal of colorectal cancer cell resistance to Fas-mediated apoptosis. International journal of cancer, 142(2), 334-346.

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Western Blot Analysis of Brown Fat Proteins

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Whole-cell extracts were prepared from tissues or brown adipocytes by homogenization in lysis buffer (29 (link)) and subjected to Western blot analysis using the following antibodies: anti–PGC-1α (28 (link)), anti-FAS, anti-SCD1, anti-actin (Santa Cruz, Dallas, TX), anti–α-tubulin, and anti-GAPDH (Abcam, Cambridge, MA).

Jun H.J., Joshi Y., Patil Y., Noland R.C, & Chang J.S. (2014). NT-PGC-1α Activation Attenuates High-Fat Diet–Induced Obesity by Enhancing Brown Fat Thermogenesis and Adipose Tissue Oxidative Metabolism. Diabetes, 63(11), 3615-3625.

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Caspase8 Activation and Fas/FADD Expression

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The activation of caspase8 and the level of protein expression of Fas and FADD, were detected by immunoblotting. Briefly, 2×10⁵ cells were transfected with pEGFPC1 vector alone or with GFP-HGV-APT. The expression of GFP and GFP-HGV-APT was confirmed by fluorescence microscopy. At the indicated time periods green cells were sorted using BD FACSAria III cell sorter (Becton Dickinson, Palo Alto CA, USA), whole cell lysates were prepared from green fluorescent cells using RIPA Buffer and according to the manufacture's instructions (Thermo-Scientific). Proteins (30 μg) were separated by denaturing SDS-PAGE and then transferred onto a PVDF membrane. The membranes were blocked in 5% non-fat dry milk in TBS 0.1% Tween and then incubated overnight with the following primary antibodies: anti-active caspase8 (Cell Signaling Technology, Beverly, USA), anti FAS, and anti FADD antibodies (Santa Cruz Biotechnology, Inc.) at 4 °C. Then, the blots were incubated with the corresponding InfraRed dye secondary antibodies (Licor). The visualization of the membrane was carried using Licor membrane scanner.

Chaabane W., Cieślar-Pobuda A., El-Gazzah M., Jain M.V., Rzeszowska-Wolny J., Rafat M., Stetefeld J., Ghavami S, & Łos M.J. (2014). Human-Gyrovirus-Apoptin Triggers Mitochondrial Death Pathway—Nur77 is Required for Apoptosis Triggering. Neoplasia (New York, N.Y.), 16(9), 679-693.

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Western Blot Analysis of Liver Proteins

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The total protein or nuclear protein from liver homogenates or cells was extracted using commercial kits (Keygen, Nanjing, China). Sample proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in a Bio-Rad Mini protean apparatus (Bio-Rad, Hercules, CA, USA) and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membranes were then blocked and incubated with primary antibodies (anti-SREBP-1, anti-ACC, anti-FAS, anti-CPT1A, anti-HADHB, anti-CROT, Santa Cruz Biotechnology, Dallas, Texas, USA) followed by incubation with a horseradish peroxidase-labeled secondary antibody (Santa Cruz Biotechnology, Dallas, Texas, USA). Densitometric analysis was performed directly from the blotted membrane using the Fusion FX5 imaging system (Vilber Lourmat, Marne, France).

Zhao L., Zhong S., Qu H., Xie Y., Cao Z., Li Q., Yang P., Varghese Z., Moorhead J.F., Chen Y, & Ruan X.Z. (2015). Chronic inflammation aggravates metabolic disorders of hepatic fatty acids in high-fat diet-induced obese mice. Scientific Reports, 5, 10222.

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Cytoplasmic Protein Extraction and Western Blot Analysis

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The cytoplasmic proteins of liver tissues and hepatocytes were extracted according to the instructions of the cytoplasmic protein extraction kit (Beyotime, Beijing, China). A total of 30–60 μg cytoplasmic protein was resolved on an 8%–12% precast gel using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Invitrogen, NY, USA) and transferred to polyvinylidene fluoride membranes (Bio-Rad, CA, USA). The membranes were then incubated with anti-p-AMPK, anti-AMPK, anti-G6Pase, anti-PEPCK, anti-ACC, anti-p-ACC, anti-FAS, anti-CPT-1a, anti-SHP1, anti-SREBP-1c (Santa Cruz, CA, USA), and anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Cell Signaling, MA, USA). Then, these membranes were washed with PBS and incubated with anti-mouse or anti-rabbit secondary antibody (Beyotime) for 1 h at room temperature. Finally, the immune complexes were developed using an enhanced chemiluminescence western blotting substrate, and protein expression levels were quantified using ImageJ software.

Yu P., Xu X., Zhang J., Xia X., Xu F., Weng J., Lai X, & Shen Y. (2019). Liraglutide Attenuates Nonalcoholic Fatty Liver Disease through Adjusting Lipid Metabolism via SHP1/AMPK Signaling Pathway. International Journal of Endocrinology, 2019, 1567095.

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Immunoblotting and Immunoprecipitation Protocols

Cited 5 times

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Western analyses and immunoprecipitations were conducted as described (3 (link), 4 (link), 16 (link), 17 (link), 19 (link), 20 (link), 27 (link)) using: anti-phospho-serine-473-Akt, anti-glyceraldehyde-phosphate dehydrogenase (GAPDH), anti-WD40/ProF, anti-PEPCK, anti-G6Pase, anti-FAS, and anti-aPKC (Santa Cruz Biotechnologies, Santa Cruz, CA); antiphospho-threonine-560/555-PKC-ζ/λ/ι (Invitrogen, Carlsbad, CA); anti-p-serine-256-FoxO1, anti-FoxO1 (Abnova, Walnut, CA); anti-PKC-λ/ι (Transduction Antibodies, Bedford, MA); anti-phospho-serine-9-GSK3β, anti-GSK3β, anti-phospho-serine-2248-mTOR, anti-mTOR, anti-ACC and mouse anti-Akt Mab (Cell Signaling Technologies, Danvers, MA); anti-IRS-1, anti-IRS-2 (Millipore, Temecula, CA); anti-SREBP (Thermo Fisher Scientific, Freemont, CA); anti-PKC-ζ (Dr. Todd C. Sacktor, State University of New York, NY); and anti-PGC-1α (Genetex, Irvine, CA), a C-terminally-directed antiserum that measures 77kDa and 91kDa PGC-1α isoforms; however, we report only on the much more abundant 77kDa isoform, which, is specifically present in human liver, and strongly influenced by FoxO1 (11 (link)).

Sajan M.P., Ivey RA I.I.I, & Farese R.V. (2015). BMI-Related Progression of Atypical PKC-dependent Aberrations in Insulin Signaling Through IRS-1, Akt, FoxO1 and PGC-1α in Livers of Obese and Type 2 Diabetic Humans. Metabolism: clinical and experimental, 64(11), 1454-1465.

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Fas/FasL Signaling Pathway Activation

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Cells were treated with increasing concentrations of poly I:C as indicated in the figure legends and lysed directly in sample buffer (0.5M Tris, pH 6.8, sodium dodecyl sulphate, bromophenol blue, glycerol, deionised water, 100mM DL-Dithiothreitol). Samples were separated on a 10% SDS-polyacrylamide gel and transferred to Immobilon-P Transfer Membrane (Merck Millipore, Billerica, MA, USA). Membranes were probed overnight at 4°C with anti-Fas (Santa Cruz Biotechnologies, CA, USA), anti-FasL, (BD Biosciences, NJ, USA), anti-β-actin (Sigma), anti-phospho-IκBα, anti-phospho-p42/44 MAPK, anti-phospho-p38 MAPK and anti-phospho-JNK specific antibody (Cell Signaling, Danvers, MA, USA). Following incubation with the appropriate secondary antibodies, membranes were developed using chemiluminescent HRP substrate (Merck-Millipore).

Lyons C., Fernandes P., Fanning L.J., Houston A, & Brint E. (2015). Engagement of Fas on Macrophages Modulates Poly I:C Induced Cytokine Production with Specific Enhancement of IP-10. PLoS ONE, 10(4), e0123635.

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Immunoblotting Analysis of Apoptosis Pathways

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Briefly, the cellular proteins were separated by SDS-PAGE and transferred onto Hybond ECL membranes (Amersham Pharmacia, Piscataway, NJ, USA). The ECL membranes were incubated with the appropriate primary antibody [anti-Fas, anti-FasL, anti-FADD, anti-JNK (Santa Cruz Biotechnology Inc.), anti-cleaved caspase-3, anti-caspase-8 (Cell Signaling Technology Inc., Danvers, MA, USA)], respectively, followed by incubation with peroxidase-conjugated secondary antibodies (Cell Signaling Technology Inc., Danvers, MA, USA). The signals were detected with the ECL system (Amersham Pharmacia). To control for lane loading, the same membranes were probed with anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Biodesign, Saco, ME, USA) after being washed with stripping buffer. The signals were quantified using a G: Box gel imaging system (Syngene, Fredrick, MD, USA).

Zhang X., Ha T., Lu C., Lam F., Liu L., Schweitzer J., Kalbfleisch J., Kao R.L., Williams D.L, & Li C. (2014). Poly (I:C) therapy decreases cerebral ischaemia/reperfusion injury viaTLR3-mediated prevention of Fas/FADD interaction. Journal of Cellular and Molecular Medicine, 19(3), 555-565.

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