Phospho-NOS3 (Ser1177) Antibody

What is Phospho-NOS3 (Ser1177) and why is it significant in research?

Phospho-NOS3 (Ser1177) refers to the endothelial nitric oxide synthase enzyme phosphorylated at serine residue 1177. This phosphorylation is physiologically significant as it represents an activated form of eNOS. The enzyme produces nitric oxide (NO), which is implicated in vascular smooth muscle relaxation through a cGMP-mediated signal transduction pathway . NO also mediates vascular endothelial growth factor (VEGF)-induced angiogenesis in coronary vessels and influences platelet activation, making this phosphorylation site critical for studying cardiovascular function, endothelial biology, and related pathologies .

What are the main applications for Phospho-NOS3 (Ser1177) antibodies?

Phospho-NOS3 (Ser1177) antibodies are primarily used in several research applications:

• Western Blot (WB): For detecting and quantifying phosphorylated eNOS in protein samples (typically at dilutions of 1:500-1:2000)

• Immunocytochemistry/Immunofluorescence (ICC/IF): For visualizing the subcellular localization of phosphorylated eNOS in cultured cells (typically at dilutions of 1:200-1:1000)

• Immunohistochemistry (IHC): For examining phosphorylated eNOS in tissue sections

• ELISA: For quantitative measurement of phosphorylated eNOS levels (at higher dilutions, e.g., 1:20000)

These applications allow researchers to examine eNOS activation status across different experimental conditions and biological systems.

What species reactivity can I expect from commercial Phospho-NOS3 (Ser1177) antibodies?

Most commercial Phospho-NOS3 (Ser1177) antibodies demonstrate confirmed reactivity with human, mouse, and rat samples . This cross-reactivity is based on the high conservation of the phosphorylation site and surrounding amino acid sequence across these species. Some antibodies may also be predicted to work with samples from additional species such as pig, bovine, horse, rabbit, and dog based on sequence homology , though these applications would typically require validation by the end user.

How is the specificity of Phospho-NOS3 (Ser1177) antibodies determined?

The specificity of these antibodies is determined through several validation methods:

• Immunogen design: Antibodies are typically raised against synthetic peptides corresponding to the region surrounding Ser1177 in human NOS3 (approximately amino acids 1144-1193)

• Affinity purification: The antibodies are purified using affinity chromatography with the specific phosphopeptide immunogen

• Functional testing: Validation in lysates from cells or tissues with known phosphorylation status (e.g., untreated vs. stimulated with factors known to induce Ser1177 phosphorylation)

• Western blot analysis: Checking for a single band of the expected molecular weight (130-160 kDa)

• Phosphatase treatment controls: Ensuring signal loss after samples are treated with phosphatases

The antibodies specifically detect endogenous levels of NOS3 protein only when phosphorylated at Ser1177, not detecting the unphosphorylated form .

How does phosphorylation at Ser1177 regulate eNOS activity compared to other post-translational modifications?

• In the absence of Ca²⁺-calmodulin, AMPK can also phosphorylate Thr-495, which has the opposite effect, inhibiting enzyme activity

• Phosphorylation at Ser-114 by CDK5 reduces eNOS activity

• Other regulatory sites include Ser-633, Ser-615, and Tyr-81

This multi-site phosphorylation creates a sophisticated control system where eNOS activity is fine-tuned by the integration of multiple signaling pathways. Researchers studying eNOS regulation must consider these interactions, as the phosphorylation status at Ser1177 alone may not fully predict enzyme activity if other inhibitory modifications are present.

What are the experimental considerations when studying eNOS phosphorylation in different cellular compartments?

eNOS localization and phosphorylation are spatially regulated within cells, presenting several important experimental considerations:

• Membrane association: eNOS can localize to plasma membrane caveolae, Golgi apparatus, and cytosolic compartments, with different phosphorylation patterns in each location

• Subcellular fractionation: When preparing samples, the fractionation method can significantly affect the detection of phosphorylated eNOS

• Fixation protocols: For immunofluorescence studies, different fixation methods may differentially preserve phospho-epitopes

• Co-localization studies: Combining phospho-eNOS staining with markers for cellular compartments (caveolin-1, Golgi markers) provides context for phosphorylation events

• Live-cell imaging: For dynamic studies, researchers should consider phospho-specific biosensors rather than fixed-cell antibody detection

When designing experiments, researchers should consider that phosphorylation at Ser1177 may occur differentially across these compartments, potentially serving distinct signaling functions based on location.

How can I differentiate between the effects of different kinases that phosphorylate eNOS at Ser1177?

Multiple kinases can phosphorylate eNOS at Ser1177, including Akt (PKB), AMPK, PKA, and CaMKII, each responding to different stimuli. To differentiate between them:

• Specific kinase inhibitors: Use selective pharmacological inhibitors (e.g., Compound C for AMPK, MK-2206 for Akt) and observe effects on Ser1177 phosphorylation

• Genetic approaches: Employ siRNA knockdown or CRISPR/Cas9 knockout of specific kinases

• Upstream pathway manipulation: Activate specific signaling pathways (e.g., insulin signaling for Akt, energy stress for AMPK)

• Temporal dynamics: Different kinases may phosphorylate Ser1177 with distinct kinetics

• Additional phosphorylation sites: Examine other phosphorylation sites that may be specifically targeted by one kinase but not others

A comprehensive approach would include phosphorylation studies under various conditions combined with manipulation of specific kinase activities to build a complete picture of the regulatory network.

What are the implications of eNOS Ser1177 phosphorylation in pathophysiological conditions?

Altered eNOS Ser1177 phosphorylation has been implicated in numerous pathophysiological conditions:

What are the optimal sample preparation protocols for detecting phospho-eNOS (Ser1177) in different applications?

Sample preparation is critical for reliable detection of phosphorylated eNOS. Here are optimized protocols for different applications:

For Western Blot:

• Rapid sample collection and immediate processing in ice-cold conditions

• Lysis buffer composition: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA

• Critical phosphatase inhibitors: 10 mM sodium fluoride, 1 mM sodium orthovanadate, 10 mM β-glycerophosphate, 1 mM sodium pyrophosphate

• Protease inhibitors: Complete protease inhibitor cocktail

• Sample storage: Aliquot and store at -80°C; avoid repeated freeze-thaw cycles

For Immunofluorescence:

• Fixation: 4% paraformaldehyde for 15 minutes at room temperature

• Avoid methanol fixation which can destroy phospho-epitopes

• Permeabilization: 0.1% Triton X-100 for 10 minutes

• Blocking: 5% BSA in PBS for 1 hour

• Primary antibody incubation: Overnight at 4°C at dilutions of 1:200-1:1000

For ELISA:

• Consistent cell number/tissue amount across samples

• Homogenization in manufacturer-recommended buffers containing phosphatase inhibitors

• Dilution optimization critical for accurate quantification

Regardless of application, phosphatase inhibitors are absolutely essential, as phospho-epitopes can be rapidly lost during sample preparation.

How should I design proper controls for phospho-eNOS (Ser1177) experiments?

Robust experimental design requires appropriate controls:

Positive Controls:

• Cells/tissues treated with agents known to induce Ser1177 phosphorylation:

  • Insulin (100 nM, 10-15 minutes)

  • VEGF (50 ng/mL, 5-10 minutes)

  • Shear stress application (12 dynes/cm², 10 minutes)

  • Statins (10 μM simvastatin, 1 hour)

Negative Controls:

• Untreated/basal condition samples

• Phosphatase treatment of positive control samples

• Preincubation of antibody with immunizing phosphopeptide

• Samples from eNOS knockout models (when available)

Specificity Controls:

• Detection of total eNOS protein (using a phosphorylation-independent antibody)

• Loading controls (β-actin, GAPDH, or vinculin)

• Phospho-null mutant (S1177A) expressing cells

Experimental Validation:

• Demonstrate expected phosphorylation changes with known pharmaceutical agents

• Show time-dependent changes in phosphorylation status

• Use two different antibody clones or detection methods as cross-validation

What are the typical troubleshooting issues when working with phospho-eNOS (Ser1177) antibodies?

Common issues and their solutions include:

Remember that phosphorylation events are highly dynamic and can change within minutes, so timing of sample collection is critical for reproducible results.

How can I quantify phospho-eNOS (Ser1177) levels accurately?

Accurate quantification of phospho-eNOS requires:

For Western Blot Densitometry:

• Always normalize phospho-eNOS signal to total eNOS from the same sample

• Calculate the phospho/total ratio rather than absolute phospho-signal

• Include standard curves with known amounts of phosphorylated protein when possible

• Ensure signal is within the linear range of detection

• Use appropriate software (ImageJ, Image Lab) with consistent analysis parameters

For Immunofluorescence Quantification:

• Maintain identical acquisition settings across all samples

• Perform Z-stack imaging to capture complete signal distribution

• Quantify mean fluorescence intensity within defined cellular regions

• Co-stain with markers to normalize to specific subcellular compartments

• Include multiple fields and cells per condition (minimum 30-50 cells)

For ELISA-based Quantification:

• Generate standard curves with each experiment

• Perform technical triplicates

• Validate antibody specificity with phosphatase-treated controls

• Consider using a capture antibody against total eNOS and detection antibody against phospho-epitope

How should I interpret changes in eNOS phosphorylation in relation to actual NO production?

While Ser1177 phosphorylation generally correlates with increased eNOS activity, the relationship with actual NO production is complex:

• Multiple regulatory factors: eNOS activity is also regulated by other post-translational modifications, cofactor availability (BH4, NADPH), substrate availability (L-arginine), and protein-protein interactions

• Phosphorylation paradox: In some pathological conditions, increased Ser1177 phosphorylation may not translate to increased NO production due to:

  • eNOS uncoupling (producing superoxide instead of NO)

  • Concurrent inhibitory modifications (Thr495 phosphorylation)

  • Cofactor deficiency

• Recommended approach for comprehensive analysis:

  • Combine phosphorylation studies with direct NO measurement techniques

  • Assess eNOS coupling status (BH4 levels, superoxide production)

  • Examine multiple eNOS regulatory sites simultaneously

  • Confirm functional outcomes of altered phosphorylation (vasodilation, angiogenesis)

Researchers should avoid equating phosphorylation status alone with functional NO production without supportive functional data.

What advanced techniques can be combined with phospho-eNOS (Ser1177) detection for more comprehensive analysis?

To gain deeper insights, researchers can combine phospho-eNOS detection with:

These advanced techniques can help unravel the complex spatial, temporal, and contextual aspects of eNOS regulation beyond simple phosphorylation status.

How can I utilize phospho-eNOS (Ser1177) antibodies in translational research applications?

Phospho-eNOS antibodies offer several translational research applications:

• Biomarker development:

  • Analysis of endothelial dysfunction in patient blood vessel samples

  • Correlation of phosphorylation status with clinical outcomes

  • Stratification of cardiovascular risk based on eNOS activity markers

• Drug development:

  • Screening compounds for effects on eNOS phosphorylation

  • Mechanism of action studies for cardiovascular therapeutics

  • Validation of on-target effects for kinase inhibitors

• Precision medicine approaches:

  • Patient-derived cell models to assess individual responses to therapies

  • Correlation of genetic variants with eNOS phosphorylation patterns

  • Personalized dosing strategies based on phosphorylation responses

• Tissue engineering:

  • Optimization of endothelial cell function in engineered vessels

  • Quality control markers for engineered tissues

  • Biomaterial effects on endothelial cell signaling

When moving to human samples, researchers should validate the antibody specificity in the specific tissue context and consider the impact of patient heterogeneity on phosphorylation patterns.

What are emerging areas of research involving phospho-eNOS (Ser1177)?

Phospho-eNOS research continues to evolve in several exciting directions:

• Single-cell analysis of eNOS phosphorylation: Understanding heterogeneity within endothelial populations

• Extracellular vesicle-associated phospho-eNOS: Exploring potential paracrine signaling mechanisms

• Mitochondrial and nuclear localized phospho-eNOS: Investigating non-canonical functions

• Computational modeling of the eNOS interactome: Predicting phosphorylation outcomes in complex signaling networks

• Development of small molecules targeting specific eNOS phosphorylation sites: More precise pharmacological manipulation

• Long-term dynamics of eNOS phosphorylation: Understanding adaptation and desensitization mechanisms

• Tissue-specific regulation of eNOS phosphorylation: Moving beyond cultured endothelial cells to intact vascular beds

• Crosstalk between eNOS phosphorylation and epigenetic regulation: Exploring longer-term consequences of altered NO signaling