Using the immunoprecipitation technique, we observed the presence of eNOS acetylation. The precipitation of proteins was achieved by utilizing an anti-eNOS antibody (Santa Cruz, Cat#sc-376,751) that was cross-linked to Protein A/G magnetic beads (MCE, HY-K0202). The proteins were detected through Western blotting using an anti-acetyl-lysine antibody (CST, Cat#9441).
Anti enos antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Anti-eNOS antibody is a tool used for the detection and analysis of endothelial nitric oxide synthase (eNOS) in various research applications. This antibody specifically recognizes the eNOS protein, allowing researchers to study its expression and distribution within biological samples.
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Lab products found in correlation
Anti β actin monoclonal antibody, by Thermo Fisher Scientific (1 mentions)
Mcdb131, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent with 4 6 diamidino 2 phenylindole, by Thermo Fisher Scientific (1 mentions)
N 6 biotinamido hexyl 3 2 pyridyldithio propionamide hpdp biotin, by Thermo Fisher Scientific (1 mentions)
Anti enos, by Santa Cruz Biotechnology (1 mentions)
Nupage 4 to 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase, by Thermo Fisher Scientific (1 mentions)
Analysis five, by Olympus (1 mentions)
Anti sqstm1 p62 antibody, by Abcam (1 mentions)
Sod and mda assay kits, by Nanjing Jiancheng (1 mentions)
Anti lc3 antibody, by Cell Signaling Technology (1 mentions)
Anti beclin 1 antibody, by Novus Biologicals (1 mentions)
Penicillin streptomycin solution p s, by ScienCell (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Anti β actin antibody, by ZSGB-BIO (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Quantione 1 d analysis software, by Bio-Rad (1 mentions)
Mouse monoclonal anti β tubulin antibody t4026, by Merck Group (1 mentions)
10 protocols using anti enos antibody
1
Immunoprecipitation of Acetylated eNOS
2
Nitric Oxide Signaling Pathway
3
Immunoprecipitation of eNOS in Myocardium
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Immunoprecipitation of the left ventricular myocardium homogenates (300 μg) was carried out using Dynabeads Protein G (Life Technologies), according to the manufacturer’s instructions. Briefly, the beads were incubated with 5 μg of anti-eNOS antibody (Santa Cruz Biotechnology) for 3 hours at 4°C. The bead–antibody complex was resuspended in PBS with 0.02% Tween. To avoid coelution of the antibody, the crosslinking reagent BS3 was added to the Dynabeads. The supernatant was removed; the samples were added to the bead–antibody complex and incubated overnight with rotation at 4°C. The Dynabead–antibody proteins were washed 3 times using PBS with 0.02% Tween. The proteins were eluted with 20 μL of elution buffer (50 mmol/L glycine, pH 2.8) by gently pipetting and incubating for 10 minutes at room temperature to dissociate the complex. The supernatants containing eluted antibody and proteins were transferred to new tubes, added to Leammli buffer, and loaded to NuPAGE 4% to 12% Bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes. The resulting blots were probed with anti-glutathione (ViroGen Corporation) and anti-eNOS (Santa Cruz Biotechnology, Inc) antibodies.
4
Quantifying Aortic Endothelial Coverage
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Immunohistochemical staining was used to quantify the percentage of the aortic lumen covered with endothelial cells. Paraffin-embedded sections (3 µm) were incubated over-night at 4°C with anti-eNOS antibody (1:20; Santa Cruz). After extensive washing steps the sections were incubated with an antirabbit biotin (Sigma, 1:500) followed by streptavidin coupled to horseradish peroxidase (1:200; Thermo Scientific) and visualized with diaminobenzidine (DAKO). The endothelial cell coverage was then quantified in percent of the lumen using imaging software (Analysis Five, Olympus, Münster, Germany).
5
Oxidized LDL-Induced Endothelial Dysfunction
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Human ox-LDL was purchased from Peking Union-Biology (Beijing, China). E64d was purchased from Sigma-Aldrich (St. Louis, MO, USA). Endothelial cell medium (ECM), fetal bovine serum (FBS), endothelial cell growth supplement (ECGS), and penicillin/streptomycin solution (P/S) were purchased from ScienCell Research Laboratories (San Diego, CA, USA). MDA and SOD assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Human sICAM-1 Quantikine ELISA Kit was purchased from R&D systems (Minneapolis, MN, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). The anti-LC3 antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-SQSTM1/p62 antibody was purchased from Abcam (Massachusetts, USA). Anti-eNOS antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-Beclin 1 antibody was purchased from Novus Biologicals (Littleton, CO, USA). Anti-Cathepsin B antibody was purchased from R&D Systems.
Anti-β-actin antibody was purchased from ZSGB-BIO (Beijing, China). Goat anti-rabbit IgG conjugated with Chromeo-546 secondary antibody was purchased from Abcam (Cambridge, UK).
Anti-β-actin antibody was purchased from ZSGB-BIO (Beijing, China). Goat anti-rabbit IgG conjugated with Chromeo-546 secondary antibody was purchased from Abcam (Cambridge, UK).
6
Nitric Oxide Synthase Immunostaining
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Mononuclear cells were separated from the bone marrow cells with lymphocyte separation medium (LSM, Capricorn Scientific, Ebsdorfergrund, Germany) according to the manufacturer’s instruction. For cytoplasmatic staining, mice bone marrow cells as well as CFU-E and BFU-E / CFU-GM colonies from methylcellulose cultures were collected onto microscope glass slides, as previously described [5 (link)]. Briefly, after fixation and incubation with the anti-neuronal NOS (nNOS, cat no. sc-5302 (1:100), Santa Cruz Biotechnology), anti-inducible NOS (iNOS, cat. no. sc-651 (1:100), Santa Cruz Biotechnology), and anti-eNOS antibodies (cat. no. sc-654 (1:100), Santa Cruz Biotechnology) immunostaining was performed using a computer-supported imaging system (analysis Pro 3.1) connected to the light microscope (Olympus AX70, Hamburg, Germany) with an objective magnification of ×40.
7
Quantification of Renal eNOS Protein
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Samples of medulla and cortex from kidney tissue containing equal amounts of protein (0.10 mg protein/lane) were separated by electrophoresis in 7.5% SDS-polyacrylamide gels, transferred to a nitrocellulose membrane (Bio-Rad, Munich, Germany), and then incubated with rabbit polyclonal anti-eNOS antibodies (1/500 dilution, epitope at the NH2 terminus, Santa Cruz Biotechnology, Santa Cruz, CA) and a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1/5,000 dilution BioRad Laboratories, USA). A marker of β-actin was used as a loading control and data were normalized to β-actin expression. Samples were revealed by chemiluminescence using an enhanced chemiluminescence reagent (Amersham Pharmacia Biotechnology, Uppsala, Sweden) for 2–4 minutes. A quantification of the bands was performed by digital image analysis using a Hewlett-Packard scanner and Totallab analyzer software (Biodynamics, Seattle, WA). All experiments were performed in triplicate [14 ].
8
Western Blot Analysis of Liver Proteins
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Liver lysates (20 µg) were electrophoresed and transferred to nitrocellulose membrane. Nitrocellulose membranes, blocked in 5% non-fat milk, 10 mmol/L Tris pH 7.5, 100 mmol/L NaCl, 0.1% Tween-20, were probed with mouse monoclonal anti-β-tubulin antibody (T4026, Sigma-Aldrich, St Louis, MO, USA) (primary antibody dilution 1∶1000), mouse monoclonal anti-MMP-9 (catalog number: IM37, Calbiochem Merck, Cambridge, MA, USA) (primary antibody dilution 1 mg/ml) and rabbit polyclonal anti-e-NOS antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (primary antibody dilution 1∶200) and then incubated in the presence of specific enzyme conjugated IgG horseradish peroxidase. Immunoreactive bands were detected by ECL detection system (Amersham Int, Buckinghamshire, UK) and analysed by densitometry. Densitometric values, expressed as Integrated Optical Intensity (IOI), were estimated in a CHEMIDOC XRS system by the QuantiOne 1-D analysis software (BIORAD, Richmond, CA, USA). Values obtained were normalized basing on densitometric values of internal β-tubulin.
9
Assessing eNOS Uncoupling in Frozen Arteries
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Rats were euthanized by exsanguination under anesthesia with thiobutabarbital, and femoral arteries were harvested and frozen in liquid N2 immediately after isolation and stored in a freezer at −80°C. To investigate the state of eNOS uncoupling, low‐temperature SDS‐PAGE was performed.20 Briefly, protein extracts were mixed with sample buffer (without β‐mercaptoethanol), and nonboiled samples were separated on SDS‐polyacrylamide gel on ice. Immunoblotting was performed with anti‐eNOS antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or with antinitrotyrosine antibodies (Santa Cruz Biotechnology).
10
Chlordecone-Induced Oxidative Stress
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Chlordecone (Sigma Aldrich St. Quentin Fallavier, France) was used at concentration found in the drinking water (2 × 10 -11 M) and in the plasma of exposed patients (5 × 10 -8 M) and dissolved in 0.1% dimethylsulfoxide cell culture medium. Superoxide dismutase (SOD) mimetic, manganese (III)tetrakis-(1-methyl-4-pyridyl)-porphyrin pentachloride (MnTMPyP), anti-β-actin antibody and inhibitors of ER (fulvestrant), NOS (nitro-L-arginine methylester, L-NAME), NADPH oxidase (apocynin), mitochondrial complexes I, III (rotenone and antimycin), mitochondrial respiration (oligomycin and antimycin A) and a decoupling agent carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP) were purchased from Sigma Aldrich (St. Quentin Fallavier, France). Human VEGF, anti-VEGF, anti-p47 phox , antiphospho-eNOS-Ser1177 and anti-eNOS antibodies were from Santa Cruz Biotechnology (Santa Cruz CA). Si-RNA anti-p47 phox and mitochondria-targeted scavenger mito-TEMPO were purchased from BD bioscience (Santa Cruz, Biotechnology). MitoNeo was gracefully provided by Prof. Richard C. Hartley and Dr. Stuart Caldwell from the School of Chemistry, University of Glasgow.
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