The phosphorylation of myocardial eNOS and nNOS was quantified by western blot (Ibrahim et al., 2014 (link); Yao and Abdel-Rahman, 2016 (link), 2017b (link)). Briefly, 80 μg of protein was separated in a 4–12% gel electrophoresis (Novex Tis-Glycine gel, Life Technologies, CA), and transferred to nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). The membranes were incubated with mouse/rabbit anti phosphor-eNOS (Ser1177, Cell Signaling, #9571,1:200) and eNOS (M221, Abcam, #ab76198, 1:500), and phosphor-nNOS (Ser1417, Thermo Fisher Scientific, Waltham, MA, #PA1–032,1:200,) and nNOS (BD Biosciences, San Jose, CA, #6100308, 1:200), the Recombinant Anti-SOD 1 (Abcam, Ann Arbor, MI, ab51254, 1:1000) and Anti- β-actin (Abcam, Ann Arbor, MI, ab8226, 1:2000) primary antibody, overnight at 4°C, and then incubated with IRDye680/800-conjugated anti-mouse/rabbit secondary antibody (LI-COR Biosciences, Lincoln,NE). To determine the monomer/dimer eNOS ratio (indicative of NOS uncoupling), the protein was separated under undenatured condition (no heating, without reducing in the sample and no antioxidant in the running buffer, running the gel under cold temperature). Odyssey Infrared Imager and Odyssey application software version 3 (LI-COR Biosciences, Lincoln, NE) were used to detect and quantify the protein bands.
Ser1177
Manufactured by Cell Signaling Technology
Sourced in United Kingdom
Ser1177 is a lab equipment product offered by Cell Signaling Technology. It is used to detect and analyze the phosphorylation of Ser1177 on the eNOS (endothelial nitric oxide synthase) protein.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Santa Cruz Biotechnology (2 mentions)
Odyssey infrared imager, by LI COR (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Odyssey application software version 3, by LI COR (1 mentions)
Anti β actin, by Abcam (1 mentions)
Novex tis glycine gel, by Thermo Fisher Scientific (1 mentions)
Irdye680 800 conjugated anti mouse rabbit secondary antibody, by LI COR (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Agilent Technologies (1 mentions)
Enos antibody, by Abcam (1 mentions)
Ab5589, by Abcam (1 mentions)
Phospho enos antibody, by Cell Signaling Technology (1 mentions)
Claudin 5, by Thermo Fisher Scientific (1 mentions)
β actin, by Abcam (1 mentions)
Cleaved caspase 3 asp175, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Trans blot turbo system, by Bio-Rad (1 mentions)
Enhanced chemiluminescence system, by Cytiva (1 mentions)
Peroxidase coupled goat anti rabbit igg, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
8 protocols using ser1177
1
Quantifying Myocardial NOS Phosphorylation
2
Western Blot Analysis of Lung Tissue
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Lung tissue was homogenized in lysis buffer (Tris–HCl 20 mM, ethylene glycol tetraacetic acid (5 mM), NaCl (150 mM), glycerophosphate (20 mM), NaF (10 mM), Triton X-100 (1%), Tween-20 (0.1%) and one tablet of protease inhibitor per 10 mL, pH adjusted to 7.5). 10 μg protein was loaded on gel (Criterion TGX gels 4–15%, cat #567-1085) and the gel was run for 1 h at 200 V, and then electrotransferred for 1 h at 100 V to nitrocellulose membranes. The membranes were blocked with 0.3% i-block in TBS-T and incubated with primary antibody overnight at 4°C, and, after washout, with horseradish-peroxidase (HRP)-conjugated secondary antibody (1:5000; Dako, Copenhagen, Denmark) for 2 h at room temperature. Excess antibody was removed by 4 times × 15 min washing, and bound antibody was detected by an enhanced chemiluminiscence kit (ECL, Amersham, United Kingdom). Protein amount was quantified using the ImageJ program (National Institutes of Health, Bethesda, MD, United States) (Bouzinova et al., 2014 (link)).
Different primary antibodies were used; e-NOS antibody (1:1.000; ab5589; Abcam, Cambridge, United Kingdom) and phospho-eNOS antibody (1:500; Ser1177; Cell Signaling Technology, Danvers, MA, United States). Pan-actin (1:1.000; #4968; Cell Signaling Technolog) served as loading reference.
Different primary antibodies were used; e-NOS antibody (1:1.000; ab5589; Abcam, Cambridge, United Kingdom) and phospho-eNOS antibody (1:500; Ser1177; Cell Signaling Technology, Danvers, MA, United States). Pan-actin (1:1.000; #4968; Cell Signaling Technolog) served as loading reference.
3
Endothelial Nitric Oxide Signaling
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Equal amounts of protein (50 μg per lane) were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels (8%–12%, Sigma-Aldrich), transferred to nitrocellulose membranes, and probed with antibodies against phospho-endothelial nitric oxide synthase (eNOS) (Ser1177, Cell Signaling; 1:500), eNOS (Becton Dickinson; 1:500), cleaved caspase-3 (Asp175, Cell Signaling, Beverly, MA, USA; 1:500), claudin-5 (Thermo Fisher Scientific; 1:500), or β-actin (Abcam, Cambridge, UK; 1:6000). The results were quantified by densitometry at n = 4 per group (ImageJ 1.34, National Institutes of Health, Bethesda, MD, USA).
4
Western Blot Analysis of Endothelial Signaling
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In a parallel experiment, endothelial cells were lysed in RIPA buffer with the addition of protease and phosphatase inhibitor cocktails (Roche). Proteins were fractionated on 4% to 12% Bis‐Tris sodium dodecyl sulfate polyacrylamide gels and transferred to nitrocellulose membranes using the Trans Blot Turbo System (BioRad). The primary antibody dilutions for Western blots were 1:1000 for rabbit anti‐human phospho‐endothelial nitric oxide synthesis (eNOS) (ser1177, Cell Signalling); 1:1000 for rabbit anti‐human eNOS; 1:1000 for rabbit (Cell Signalling), 1:1000 for rabbit anti‐human AKT2 (Cell Signalling CST‐4691S); and 1:2000 for rabbit anti‐human phospho‐AKT2 Ser473 (Cell Signalling CST‐4060S). The membranes were then incubated with swine anti‐rabbit horseradish peroxidase–linked IgG antibody (1:3000; Dako, P0399) or rabbit anti‐goat horseradish peroxidase–linked IgG antibody (1:3000; DAKO, P0160) followed by detection of the proteins using femto ECL chemiluminescent horseradish peroxidase substrate (Thermo Scientific) and the ChemiDoc XRS system (BioRad). β‐Actin (rabbit anti‐human actin; Abcam, ab8227; 1:5000) was used as loading control.
5
Western Blot Analysis of eNOS in Organs
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Standard Western blotting analysis was performed in lung and cardiac lysates [14 (link)] using antibodies against p-eNOS (Ser1177, 1:1000, Cell Signaling Technology) and e-NOS (1:1000, Santa Cruz Biotechnology, Santa Cruz). Secondary HRP-conjugated antibodies were applied for 1 h at room temperature, and signals were detected by using the enhanced chemiluminescence system (Amersham, Arlington Heights, IL, USA) of a commercial ECL kit. Quantification of the band intensities was performed using NIH ImageJ.
Blood hemoglobin (Hb, Drabkin’s reagent) and plasma nitrates and nitrites (NOx, colorimetric Griess reaction) were measured in heparinized samples taken after euthanasia.
Blood hemoglobin (Hb, Drabkin’s reagent) and plasma nitrates and nitrites (NOx, colorimetric Griess reaction) were measured in heparinized samples taken after euthanasia.
6
Western Blot Analysis of Signaling Proteins
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Cell lysates (30 μg of each protein sample) were loaded onto sodium dodecyl sulfate polyacrylamide gel electrophoresis and, thereafter, they were transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories), which were incubated with specific primary antibodies: anti phospho-eNOS (1:1000; Ser1177, Cell Signaling Technologies), anti eNOS (1:1000; Cell Signaling Technologies), anti iNOS (1:500; Santa Cruz Biotechnology), anti phospho-Akt (1:1000; Ser 473, Santa Cruz Biotechnology), anti Akt (1:1000; Santa Cruz Biotechnology) anti phospho-ERK 1/2 (1:1000; Thr 202/Tyr 204, Santa Cruz Biotechnology), anti ERK 1/2 (1:1000; Santa Cruz Biotechnology), anti Cleaved Caspase-9 (1:1000; Abcam, Cambridge, UK) and anti Cytochrome C (1:1000, Santa Cruz Biotechnology). The membranes were washed and incubated with horseradish peroxidase-coupled goat anti-rabbit IgG (Sigma), peroxidase-coupled rabbit anti-goat IgG and horseradish peroxidase-coupled goat anti-mouse IgG (Sigma) for 45 min and were developed through Western Lightning Chemiluminescence (PerkinElmer Life and Analytical Sciences). Protein expression was calculated as a ratio towards specific total protein expression or β-actin (1:5000; Santa Cruz Biotechnology) detection.
7
Western Blot Analysis of Renal Proteins
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Renal tissues were homogenized with lysis buffer containing 1 mmol/l PMSF, 10 μg/ml aprotinin, and 10 μg/ml leupeptin. After the homogenization, an aliquot of supernatant was taken for protein measurement with Bio-Rad protein assay. Thirty micrograms of protein was separated by SDS–PAGE and transferred to a nitrocellulose membrane. The membranes were incubated with blocking solution (5% milk added to TBST buffer) at room temperature for 1 h, then incubated with primary antibodies against phospho-endothelial nitric oxide synthesis (eNOS, Ser-1177, Cell signaling), NADPH oxidase subunits gp91phox and p22phox, tumor necrosis factor (TNF) α, interleukin (IL)1β, transforming growth factor (TGF) β1, and fibronectin (Santa Cruz Biotechnology Inc.) at 4°C overnight (all these primary antibodies were diluted by blocking solution in 1:500) and washed three times with TBST containing Tween 20, then the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:2000 dilution using blocking solution) for 1 h at room temperature. The signals were detected by ECL using hyperfilm and ECL reagent (Santa Cruz Biotechnology Inc.). The membranes were reblotted for β-actin (1:500 dilution, Santa Cruz Biotechnology Inc.), to serve as a loading control. The data were normalized to β-actin and expressed as fold change versus control group.
8
HDL-Stimulated NO Production in ECs
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HDLs were incubated for 30 min in primary cultures of human umbilical vein ECs (Clonetics, Lonza) at a protein concentration of 1.0 mg of protein per milliliter. After incubation, NO production was measured using a diacetate derivative of 4,5-diaminofluorescein (Sigma-Aldrich Chemie) as previously described. 13 (link) Fluorescence intensity was detected using a Synergy Multi-Mode microplate reader equipped with the GEN5 software (BioTek), and data were normalized by total protein concentration in the cell lysate. To assess if NO production was mediated by eNOS, eNOS activation by phosphorylation was evaluated by SDS-PAGE and immunoblotting after 10 min of incubation with HDL. Membranes were developed against phosphorylated eNOS (Ser1177, Cell Signalling Technology), stripped, and reprobed with an antibody against total eNOS. Bands were visualized by enhanced chemiluminescence (GE Healthcare Biosciences) and analyzed with a GS-690 Imaging Densitometer and Multi-Analyst software (Bio-Rad Laboratories) as described. 23 (link)
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