Protein was extracted using radioimmunoprecipitation assay buffer supplemented with 1 mM phenylmethylsulfonyl fluoride (Sigma, St. Louis, MO). After the protein concentration was measured with a bicinchoninic acid (BCA) protein assay kit (Beyotime, China), the proteins were separated in 10% or 12% (w/v) sodium dodecyl sulfate polyacrylamide gels and then transferred to a 0.22 μm pore size PVDF membrane (Merck Millipore). After blocked with TBST containing 5% (w/v) bovine serum albumin, the membranes were incubated with primary antibodies (anti-SIRT1: diluted at 1 : 1000, Cell Signaling Technology, USA; anti-LC3B, anti-P62, and anti-cleaved-caspase 3: diluted at 1 : 1000, Abcam, USA; anti-Bax, anti-Bcl-2, anti-P65/NF-κB, anti-p-P65/p-NF-κB, and anti-β-actin: diluted at 1 : 000, Proteintech, China) overnight at 4°C. The membranes were washed three times with cold TBST for 15 min and then incubated with horseradish peroxidase-labeled secondary antibody (Beyotime, Wuhan, China) at room temperature for 1 h. Finally, protein blots were detected via chemiluminescence (ECL-Kit, Bio–Rad, Cambridge, MA, USA) and quantified using Quantity One analysis software, version 4.4 (Bio–Rad).
Anti sirt1
Manufactured by Cell Signaling Technology
Sourced in United States, China
Anti-SIRT1 is a polyclonal antibody that specifically recognizes the SIRT1 (Sirtuin-1) protein. SIRT1 is a NAD+-dependent deacetylase that plays a role in a variety of cellular processes, including metabolism, cell survival, and gene expression regulation.
Automatically generated - may contain errors
Lab products found in correlation
Anti ampk, by Cell Signaling Technology (16 mentions)
Anti bcl 2, by Cell Signaling Technology (14 mentions)
Anti gapdh, by Cell Signaling Technology (13 mentions)
Anti bax, by Cell Signaling Technology (13 mentions)
Anti β actin, by Cell Signaling Technology (10 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (9 mentions)
Anti p53, by Cell Signaling Technology (8 mentions)
Pvdf membrane, by Merck Group (8 mentions)
Anti parp, by Cell Signaling Technology (7 mentions)
Anti p ampk, by Cell Signaling Technology (7 mentions)
Anti β actin, by Merck Group (7 mentions)
Anti acetyl p53, by Cell Signaling Technology (6 mentions)
Anti bak, by Cell Signaling Technology (6 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (6 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Anti mtor, by Cell Signaling Technology (5 mentions)
Anti lc3b, by Cell Signaling Technology (5 mentions)
Anti sirt3, by Cell Signaling Technology (5 mentions)
Ripa lysis buffer, by Beyotime (5 mentions)
Anti acetylated lysine, by Cell Signaling Technology (5 mentions)
102 protocols using anti sirt1
1
Western Blot Analysis of Protein Signaling
2
Western Blot Analysis of AMPK Signaling
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Cells were lysed using ice-cold lysis buffer (0.1 mL of 50 mM Tris-HCl (pH 7.2) containing 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, and 1% NP-40), and the so-obtained cell lysates were assayed for protein concentration by Bradford staining. Equal amounts of protein (20 μg/mL) were electrophoresed on 10% SDS-acrylamide gels and transferred to nitrocellulose membranes using an electric transfer system. Nonspecific binding was blocked by treating membranes with 3% skim milk in TBST buffer (5 mM Tris-HCl, pH 7.6, 136 mM NaCl, and 0.1% Tween-20) for 1 h. Blots were incubated for 1 h at room temperature with primary antibody against anti-phospho-AMPKα (Thr 172), anti-AMPKα, anti-phospho-acetyl-CoA carboxylase (ACC) (Ser79), anti-ACC, anti-SIRT1 (Cell Signaling Technology, Danvers, MA, USA), anti-PGC1α (Santa Cruz Biotechnology, Santa Cruz, CA, USA), glucose transporter (GLUT) 4 (Santa Cruz Biotechnology), and anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA) and then incubated for 1 h at RT with horseradish peroxidase- (HRP-) labeled anti-mouse IgG (1 : 1000, Santa Cruz Biotechnology), washed with 1x TBST three times, and developed with the ECL Western detection reagents (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Protein bands were quantified by densitometry using Image J.
3
Protein Modification and Regulation Assays
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NButGT, Ac-5SGlcNAc were provided by D. Vocadlo (Simon Fraser University)(48 (link)). SRT1720, EX-527, Compound C purchased from Selleck-Chemicals (Houston, TX). MG132 purchased from Sigma-Aldrich (St. Louis, MO). pcDNA3.1-SIRT1-Flag was gift from E. Verdin (Addgene plasmid#: 13812). pLenti4-HA-OGT (provided by K. Vosseller, Drexel University). Antibodies used: anti-actin, anti-FOXM1 and anti-RL2 from Santa Cruz Biotechnology; pERK1/2, anti-OGT and anti-O-GlcNAc from Sigma-Aldrich; anti-acetylated p53(K382), anti-AMPK, anti-pAMPK(S172), anti-ERK1/2, anti-MMP2, anti-pRaptor(792), anti-Raptor, anti-SIRT1 and anti-Ubiquitin(K48) from Cell Signaling (Danvers, MA); anti-Cdh1 and anti-p53 from Neobiolabs; anti-MMP9 from Novus-Biologicals (Littleton, CO); integrin α5 and α6 are from BD-Pharmingen (San Jose, CA).
4
Western Blot Analysis of Apoptosis Markers
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Fetal bovine serum (FBS) and penicillin/streptomycin were obtained from Gibco/BRL Life Technologies (Grand Island, NY, USA). The anti-Bak, anti-PARP, anti-Bcl-2, anti-p53, anti-phospho-p53, anti-acetyl-p53, anti-SIRT1, and anti-cyclin D1 antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). The anti-b-actin antibody was from Millipore Corp. (Temecula, CA, USA). The antisera to tNOX used in Western blot analyses were generated as described previously [22 (link)]. The 3,3′-Dihexyloxacarbocyanine iodide [DiOC6(3)] was purchased from Calbiochem (San Diego, CA, USA). The anti-mouse and anti-rabbit IgG antibodies and other chemicals were purchased from the Sigma Chemical Company (St. Louis, MO, USA), unless specified otherwise.
5
Western Blot Analysis of SIRT1 Protein
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The frozen CeA tissues were homogenized in ice-cold RIPA lysis buffer containing protease inhibitors and phenylmethylsulfonyl fluoride (Beyotime Biotech, Jiangsu, China). The supernatants were collected after centrifugation at 12,000 g for 15 min at 4°C, and the protein concentration was determined using the BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, United States). Equal amounts of protein samples were separated by SDS-polyacrylamide gel electrophoresis (Beyotime Institute of Biotechnology, China) and transferred onto a nitrocellulose membrane. Then, the membrane was incubated with the following primary antibodies at 4°C overnight: anti-SIRT1 (Cell Signaling Technology, Beverly, MA, United States), and anti-β-actin (Bioworld, Louis Park, MN, United States), followed by incubation with the IRDye 800CW second antibody (Li-Cor, Lincoln, NE, United States). The immunoreactive bands were detected using an Infrared Imaging System (Gene Company Limited, Hong Kong, China) and analyzed with ImageJ software.
6
Sirt1 and HIF-1α Immunoprecipitation
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Approximately 1 mg of total proteins was incubated overnight at 4°C with anti‐Sirt1 antibody (Cell Signaling) or anti‐HIF‐1α antibody (Novus Biologicals, Littleton, CO, USA) followed by precipitation with 20 µl of protein A/G‐Plus‐Agarose (Santa Cruz, Dallas, TX, USA) for 4 hr at 4°C. The precipitated complexes were immunoblotted with anti‐Sirt1 (Cell Signaling), anti‐HIF‐1α (Novus Biologicals), or anti‐acetyl‐lysine (Cell Signaling).
7
Visualizing SIRT1 and DNA Damage in HPDL Cells
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HPDL cells were plated on fibronectin-coated glass coverslips and cultured for 24 hours. Cell layers were fixed in 4% PFA for 10 min, permeabilized with Triton X-100 for 10 min, and blocked with 1.5% BSA for 1 hour. Actin fibers were stained with Anti stain 555 phalloidin or Anti stain 555 phalloidin (Cytoskeleton, CO, USA). Anti-SIRT1 (Cell Signaling Technology; CST, MA, USA) and γH2AX (CST) antibodies were used as primary antibodies and Alexa Fluor 594 goat anti-rabbit IgG (CST) was used as the secondary antibody for ICC staining. Nuclei were stained with VECTASHIELD Mounting Medium with 4'6-diamidino-2-phenylindole (DAPI) (Vector Lab., CA, USA). Immunofluorescence and quantitative image analysis were performed under a Leica SP8 microscope (Leica) using 63× or 100× oil immersion lenses with a numerical aperture (NA) of 1.4. After acquisition, images were processed with Airyscan (Zen software; Carl Zeiss, Oberkochen, Germany).
8
Evaluating Endothelial Function Biomarkers
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HUVECs were homogenized in RIPA lysis buffer (CWBIO, Jiangsu, China, #CW2333) containing a proteinase and phosphatase inhibitor cocktail. Equal protein quantities were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electro-transferred onto polyvinylidene fluoride membranes. The blots were blocked in 5% fat-free milk and probed with primary antibodies against p-eNOS, eNOS, and SIRT1 (1:1000; Cell Signaling Technologies, Inc., Danvers, MA, USA, #9570, #32027, and #9475) and p53, p21, p16, and β-actin (1:5000; Proteintech, #10442–1-AP, #10355–1-AP; and 1:1000; Proteintech, #16717-1-AP). Blots were incubated overnight (12 h) at 4 °C with primary antibodies, blocked with 5% bovine serum albumin (for phosphorylation-specific antibodies) or 5% skim milk (for other antibodies) at room temperature (20–25 °C) for 2 h, and incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000; Proteintech). Antibody labeling was visualized using an electrochemiluminescence system (Bio-Rad, Hercules, CA, USA). β-Actin was used as loading controls. Immunoprecipitation was applied to isolate eNOS and quantify its acetylation as described previously [24 ]. Anti-SIRT1 (1:1000; Cell Signaling Technologies, Inc., #9475) and anti-acetylated lysine antibodies (1:1000; Cell Signaling Technologies, Inc., #9441) were used.
9
Western Blot Analysis of DNA Damage Signaling
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Whole cell protein extracts were prepared according to Laemmli (1970 (link)). The primary antibodies used were: anti-ATM (1:500), anti-phospho-ATM Ser1981 (1:500), anti-p300 (1:500) (Abcam, Cambridge, UK), anti-GAPDH (1:50000) (Millipore, Darmstadt, Germany); anti-p21WAF1/Cip1 (1:500) (Sigma-Aldrich, St. Louis, USA); anti-p53 (1:500), (Santa Cruz Biotechnology, Santa Cruz, USA); anti-ATR (1:500), anti-phospho-ATR Ser428 (1:500), anti-phospho-p53 Ser15 (1:250), anti-acetyl-p53 Lys382 (1:200), anti-SIRT1 (1:250), anti-phospho-SIRT1 Ser47 (1:250), anti-p38 MAPK (1:500), anti-phospho-p38 MAPK Thr180/Tyr182 (1:500), anti-phospho-MAPKAPK-2 Thr334 (1:500), anti-AMPKα (1:500), anti-phospho-AMPKα Thr172 (1:1000), anti-ACC (1:500), anti-phospho-ACC Ser79 (1:1000), anti-mTOR (1:500), anti-phospho-mTOR Ser2448 (1:500), anti-phospho-S6 Ser235/236 (1:1000) (Cell Signaling Technology, Denvers, USA), anti-Rb (1:250) (NeoMarkers, Fremont, USA). The respective proteins were detected after incubation with one of the horseradish peroxidase-conjugated secondary antibodies (1:2000) (Dako, Glostrup, Denmark), using an ECL system (Thermo Scientific, Rockford, USA), according to the manufacturer’s instructions.
10
Western Blot Analysis of Metabolism Proteins
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Tissues and Cultured cells were lysed with RIPA buffer supplied with phosphatase and protease inhibitor cocktail (Beyotime Biotechnology, China). Concentrations of protein were determined by the bicinchoninic acid (BCA) assay (Beyotime Biotechnology, China). Total protein extract (40 μg) was separated by 10% SDS polyacrylamide gel electrophoresis and transferred to PVDF membranes (BioRad, America). The primary antibodies were as follows: anti-PPARα, anti-PPARδ, anti-AMPK, anti-p-AMPK, anti-SIRT1, and anti-IκBα (1:1,000; Cell Signalling Technology, Abcam). Immunoreactive bands were quantitatively analyzed using ImageJ software.
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