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Total enos

Manufactured by Cell Signaling Technology
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The Total eNOS assay kit is a quantitative enzyme-linked immunosorbent assay (ELISA) designed to measure the total levels of endothelial nitric oxide synthase (eNOS) protein in cell and tissue lysates. eNOS is a key enzyme involved in the production of nitric oxide, which plays a crucial role in various physiological processes.

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18 protocols using total enos

1

Western Blot Analysis of Phosphorylated eNOS and Akt

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The rat MAB was homogenized in a lysis buffer composed of 50 mmol/L Tris-HCl (pH
7.4), NP-40 (1%), sodium deoxycholate (0.5%) and SDS (0.1%). Samples were
centrifuged at 5,000 × g for 10 min (4ºC). Forty micrograms of protein
were separated by electrophoresis on a 10% polyacrylamide gel, and transferred
onto a nitrocellulose membrane. Skimmed milk 5% diluted in Tris-buffered saline
solution with Tween 20 was used to block nonspecific binding sites (1 h at
24ºC). Membranes were then incubated overnight at 4ºC with the following primary
antibodies: p-eNOS (Ser1177) (diluted 1:1000, 9571, Cell Signaling,
Danvers, MA, USA), p-eNOS (Thr495) (diluted 1:1000, 9574, Cell
Signaling), total eNOS (diluted 1:1000, 9572, Cell Signaling), P-protein kinase
B (P-Akt) (Ser473) (diluted 1:1000, 4058, Cell Signaling) and total
Akt (diluted 1:1000, 9272, Cell Signaling). Membranes were then incubated with
secondary antibodies for 90 min at room temperature. Signals were revealed with
chemiluminescence and quantified densitometrically. The results are expressed as
the non-phospho/total proteins ratio.
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2

Oxidized LDL Induced Endothelial Dysfunction

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Growth factor-reduced Matrigel matrix was purchased from Corning (Bedford, MA, USA). FxCycle™ PI/RNase Staining Solution, H2DCFDA, DAF-FM diacetate, and DAPI were bought from Life Technologies Corporation (Eugene, OR, USA). Anhydrous sodium sulfide (Na2S), Pierce BCA protein assay kit, RIPA buffer, protease and phosphatase inhibitor mini tablets, RevertAid RT Reverse Transcription Kit, PowerUp SYBR™ Green Master Mix, TRIzol Reagent and CD68 antibody were procured from Thermo Scientific (Rockford, IL, USA). Na2S solution was prepared in molecular biology grade water. Human oxidized low-density lipoprotein (oxLDL) was acquired from Kalen Biomedical, LLC (Montgomery, MD, USA). Cell proliferation reagent WST-1 was obtained from Roche Diagnostics GmbH (Mannheim, Germany). Phospho-eNOS (Ser1177), phospho-AKT (Ser473), phospho-ERK1/2 (Thr202/Tyr204), total AKT, total ERK1/2, total eNOS, Ki67, and CD45 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). GAPDH antibody was procured from Santa Cruz Biotechnology (Dallas, TX, USA). LYVE-1 antibody was purchased from Abcam (Cambridge, MA, USA). CD31 antibody was obtained from R&D Systems, Inc. (Minneapolis, MN, USA). Ready-to-use intercept blocking buffer to block the nitrocellulose membranes was purchased from Li-Cor Biosciences (Lincoln, NE, USA).
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3

Immunohistochemical Analysis of Vascular Markers

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Tissue sections were heated in Tris-EDTA buffer to retrieve antigen epitopes, blocked by 10% normal goat serum and Avidin/Biotin blocking reagent (Vector Laboratories, Burlingame, CA, USA) and stained with the following primary antibodies at 4 °C overnight: anti-MAGI1 (Sigma-Aldrich, Buchs, Switzerland, cat. no. HPA031853), P-eNOS (Ser1177, Cell Signaling, Danvers, MA, USA; cat. no. 9571S) and total eNOS (Cell Signaling, Danvers, MA, USA; cat. no. 9572S). Sections were incubated with biotinylated secondary antibodies followed by Vectastain ABC Kit (Vector Laboratories, Burlingame, CA, USA) with DAB peroxidase substrate (Sigma-Aldrich). Sections were counterstained with haematoxylin before mounting.
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4

Western Blot Analysis of Vascular Proteins

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Total protein was extracted from mesentery beds. Frozen tissues were homogenized in 50 mmol/L Tris/HCl (pH 7.4) lysis buffer (containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA, 0.1% SDS, 2 mmol/L Na3VO4, 1 mmol/L PMSF, 1 μg/mL pepstatin A, 1 μg/mL leupeptin, and 1 μg/mL aprotinin). Total protein extracts were cleared by centrifugation at 12,000 g for 10 min and the pellet was discarded. Proteins from homogenates of vascular tissues (50 μg) were separated by electrophoresis on a polyacrylamide gel (10%) and transferred on to a nitrocellulose membrane. Non-specific binding sites were blocked with 5% skim milk or 1% BSA in Tris-buffered saline solution with Tween 1% at 24°C. Membranes were then incubated with specific antibodies overnight at 4°C. Antibodies were as follows: Ser1177- endothelial Nitric Oxide Synthase (eNOS) and total eNOS (Cell Signaling, 1:500), soluble guanylyl cyclase (sGC) α and β subunits (Abcam, 1:500), Superoxide Dismutase 1 (SOD1), SOD2 and Catalase. Antibody to β-actin (Sigma) was used as internal housekeeping control. After incubation with secondary antibodies, signals were revealed with chemiluminescence, visualized by autoradiography and quantified densitometrically.
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5

Antibody-based Angiogenesis Assay Protocol

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Antibodies used were cMyc (#5605, 1:1000), KLF4 (#4038, 1:1000), Sirt1 (#8469, 1:1000), β-Actin (13E5, #4970, 1:4000), β-Actin (8H10D10, #3700, 1:4000) and total eNOS (#32027, 1:1000) from Cell Signaling (Danvers, MA, USA). Phosphorylated eNOS (p-eNOS) detecting serine 1177 (ab215717, 1:500), anti-glutathione peroxidase-1 antibody (ab108427, 1:1000), SPRED1 antibody [M23-P2G3] (ab64740, 1:1000) and anti-Ki67 antibody (ab15580, 1:300) were from Abcam (Cambridge, MA, USA). CD47 MIAP301 (sc-12731, 1:500) and CD47 B6H12 (sc-12730, 1:500) were from Santa Cruz Biotech (Santa Cruz, CA, USA). Matrigel® Growth Factor Reduced (GFR, Product Number 354230) was from Corning, Inc. (Corning, NY, USA). Endothelial cell growth media (Catalog #: CC-3156) was from Lonza (Basel, Switzerland). VEGF Recombinant Human Protein (#PHC9393) was from ThermoFisher Scientific (Waltham, MA, USA) and reconstituted in PBS according to the manufacturer’s instructions. Dispase® II was from Sigma-Aldrich (St. Louis, MI, USA). Mouse FibrOut™ 11 and Human FibrOut™ custom prepared for Blood Vessels and Endothelial Tissues were from Chi Scientific Inc. (Maynard, MA, USA).
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6

Protein Expression Analysis in Renal Cortex

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The total proteins of the renal cortical tissues were extracted with a Pro-Prep Protein Extraction Solution (Intron Biotechnology, Gyeonggi-Do, Korea), following the manufacturer instructions. Western blot was performed with specific antibodies for CaSR (Thermo Fisher Scientific Inc, Waltham, MA), CaMKKα/β (Santa Cruz Biotechnology), PGC-1α (Novus Biologicals, Littleton, CO), total LKB1(Cell Signaling Technology, Danvers, MA), phospho-Ser428 LKB1(Cell Signaling Technology), total AMPK (Cell Signaling Technology), phospho-Thr172 AMPK (Cell Signaling Technology), total eNOS (Cell Signaling Technology), phospho-Ser1177 eNOS (Cell Signaling Technology), B cell leukemia/lymphoma 2 (BCL-2), BCL-2-associated X protein (BAX) (Santa Cruz Biotechnology), Cu/Zn superoxide dismutase (SOD1) (Assay Designs, Ann Arbor, MI), Mn superoxide dismutase (SOD2) (Abcam), beclin-1 (Novus Biolobicals), LC3B (Sigma-Aldrich), tumor necrosis factor-α (Abcam), interleukin-1β (IL-1β) (Abcam), and β-actin (Sigma-Aldrich). After incubation with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (secondary antibody) (Cell Signaling Technology), target proteins were visualized by an enhanced chemiluminescence substrate (ECL Plus; GE. HealthcareBio-Science, Piscataway, NJ).
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7

Quantifying eNOS Expression in Aortic Tissue

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Formalin-fixed, paraffin-embedded aortic tissue was section into 1-μm slices and then immunolabeled with either total eNOS (1:30; Cell Signaling Technology) or phosphorylated eNOS (1:50; Abcam). Primary antibodies were visualized with fluorescent conjugated secondary antibodies (goat anti-mouse-594, or goat anti-rabbit-647). Tissue sections were counterstained with DAPI. Images were taken with a laser scanning confocal microscope (Zeiss LSM, Plan Apochromat 40 × 1.3 oil objective). For both stains, 10 regions of interest were captured from each patient. Total eNOS and phosphorylated eNOS were measured by fluorescence intensity with ImageJ (National Institutes of Health, Bethesda, Md).
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8

Western Blot Analysis of Akt and eNOS

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Equal amounts of proteins (40 μg total protein or 80 μg nuclear protein per lane) were loaded onto a 12.5% gradient 2-amino-2-(hydroxymethyl)propane-1,3-diol hydrochloride (Tris–HCl) sodium dodecyl sulfate–polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane. Nonspecific binding to the membrane was blocked for 1 h at room temperature with 5% nonfat milk in 1 × TBS, followed by incubation with primary antibodies against total Akt (rabbit monoclonal 1 :1000; Cell Signaling Technology, Danvers, MA), p-Akt (Ser473, rabbit monoclonal 1:2,000; Cell Signaling Technology), total eNOS (rabbit monoclonal 1:1000; 1:250 dilution, Cell Signaling, USA), and p-eNOS (Ser1179, 1:250 dilution, Invitrogen, USA) overnight at 4 °C. After washing with TBST three times, membranes were incubated with horseradish peroxidase-conjugated rabbit or goat secondary antibody (1:10,000 dilution; Kang Chen Biotechnology, Guangzhou, China) for 1 h at room temperature, followed by three washes for 10 min each. Blots were developed using enhanced chemiluminescent reagents (Thermo Fisher Scientific, Pittsburgh, PA, USA) and target band density was scanned using an LAS-3000 detection system. Image J software was used to analyze band intensities.
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9

Compound 1213 Modulates Endothelial eNOS

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Compound 1213 was added to EC culture immediately before hypoxia exposure. One hour later, phosphorylation of serine‐1177 endothelial nitric oxide synthetase (eNOS) was measured by Western blot in both ECs incubated in room air (RA) or exposed to hypoxia. Commercially available antibodies for serine‐1177 PO4 eNOS, total eNOS (Cell Signaling Technologies Danver, MA), and secondary antibodies (Santa Cruz Biotechnologies, Santa Cruz, CA) were used. Cells were washed with ice‐cold PBS twice and were immediately lysed in the plate using RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Roche Applied Science, Indianapolis, IN). Proteins were resolved on SDS‐PAGE and blotted on PVDF membrane. Beta‐actin (Ab from Genscript, Piscataway, NJ) was used as a loading control for normalization. Different doses of C1213 were used to assess target dose in relation to maximum physiological function and 10 μM was selected for studies. Densitometry was performed using Image J software which is freely available at https://imagej.nih.gov/ij/index.html. cGMP as a product of NO production was also assayed in EC culture cells after treating them with vehicle or C1213 (ELIZA assay, Cayman, Ann Arbor, Michigan).
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10

Western Blot Analysis of Protein Expression

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Protein samples with equal amount of total protein (20 μg) were separated on SDS-PAGE (8–15%). The separated protein gel was then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk at room temperature for 1 h in Tris-buffered saline containing 0.1% Tween-20, primary antibody incubation (TXNIP, Cat#: 14715; Thioredoxin 1, Cat#: 2285; GLUT4, Cat#: 2213; pyruvate dehydrogenase E1-alpha [PDH E1α], Cat#: 31866; p-eNOS, Cat#: 9571; Total-eNOS, Cat#: 5880; NLRP3, Cat#: 13158; VCAM-1, Cat#: 13662; ICAM-1, Cat#: 4915; Cleaved-IL-1β, Cat#: 83186 and GAPDH, Cat#: 5174 were all from Cell Signaling Technology; Anti-Nitro tyrosine antibody [Cat#: ab42789, Abcam Company]) was carried out overnight at 4°C. Afterward, secondary antibody incubation with a peroxidase-conjugated AffiniPure goat anti-rabbit or anti-mouse IgG was conducted for 90 min at room temperature. After washing 3 times, the membranes were subjected to ECL detection. Densitometric analysis was performed using the Tanon Gel Imaging System (Shanghai Tanon, Shanghai, China). The housekeeping gene GAPDH served as a loading control.
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