Phospho-ALOX5 (Ser523) Antibody

What is Phospho-ALOX5 (Ser523) Antibody and what does it detect?

Phospho-ALOX5 (Ser523) antibody is a phospho-specific polyclonal antibody that recognizes the ~80 kDa 5-lipoxygenase (5-LO) protein exclusively when it is phosphorylated at the serine 523 residue. The antibody is typically produced in rabbits using synthetic phosphopeptides corresponding to amino acid residues surrounding Ser523 of human 5-LO conjugated to keyhole limpet hemocyanin (KLH) . This antibody demonstrates specificity across human, mouse, rat, and non-human primate samples due to the 100% sequence homology in this region .

The phospho-specificity of the antibody can be validated experimentally, as immunolabeling is completely eliminated by treatment with lambda phosphatase (λ-phosphatase), confirming its selective binding to the phosphorylated form of the protein .

How does phosphorylation at Ser523 affect ALOX5 function?

Phosphorylation at Ser523 by Protein Kinase A (PKA) plays an inhibitory role in ALOX5 function, contrasting with phosphorylation at other sites:

Specifically, PKA-mediated phosphorylation at Ser523 inhibits ALOX5 translocation from the cytoplasm to the nuclear membrane, thereby reducing its enzymatic activity and subsequent leukotriene synthesis . This inhibitory mechanism is physiologically significant as it may serve as a natural brake on inflammatory processes .

Research has demonstrated that replacement of Ser523 with alanine or glutamate leads to increased activity of ALOX5, further confirming the inhibitory effect of phosphorylation at this position .

What experimental techniques can be used with Phospho-ALOX5 (Ser523) Antibody?

The Phospho-ALOX5 (Ser523) antibody is primarily optimized for Western blot applications, with recommended dilution of 1:1000 for most tissue samples, particularly brain tissue . The following methodological approaches can be implemented:

• Western Blotting:

  • Detects the ~80 kDa doublet of 5-LO phosphorylated at Ser523

  • Optimal results in rat hippocampal or brain cortex lysates

  • Standard blocking with 5% BSA in TBST recommended

  • Secondary antibody: Anti-rabbit IgG

• Phosphorylation Validation:

  • Lambda phosphatase treatment (1200 units for 30 minutes) to confirm phospho-specificity

  • Parallel samples: untreated vs. phosphatase-treated

• Subcellular Localization:

  • Differential centrifugation followed by Western blot to track translocation

  • Cytosolic vs. nuclear/membrane fractions analysis

• Protein-Protein Interaction:

  • Co-immunoprecipitation to investigate interactions with PKA, cPLA2, or COX2

  • Proximity ligation assays to visualize protein interactions in situ

While immunohistochemistry applications are not prominently mentioned in the literature for this specific antibody, optimization for this technique may be possible with appropriate positive controls and validation steps .

What is the functional significance of ALOX5 Ser523 phosphorylation in inflammation?

ALOX5 Ser523 phosphorylation plays a crucial role in regulating inflammatory processes through several mechanisms:

• Inflammation Limitation: Under normal physiological conditions, PKA-mediated phosphorylation at Ser523 serves as a regulatory mechanism to limit excessive inflammation by inhibiting leukotriene synthesis .

• Pathway Selection: Phosphorylation at Ser523 determines whether ALOX5 interacts with cytosolic phospholipase A2 (cPLA2) to produce pro-inflammatory leukotriene B4 or with cyclooxygenase-2 (COX2) to produce anti-inflammatory 15-epilipoxin-A4 (15-epi-LXA4) .

• Disease Relevance: Abnormal signaling through cAMP and PKA pathways affecting Ser523 phosphorylation may contribute to various inflammatory diseases, including asthma, fibrosis, and atherosclerosis .

• Therapeutic Targeting: Medications such as atorvastatin and pioglitazone can modulate the production of inflammatory mediators through mechanisms potentially involving ALOX5 phosphorylation, highlighting the pathway as a therapeutic target .

This regulatory mechanism represents a potential point of intervention for anti-inflammatory therapies, making the phospho-specific antibody a valuable tool for studying the role of 5-LO regulation in various disease contexts .

How can researchers validate the phospho-specificity of anti-ALOX5 (Ser523) antibodies in their experiments?

Rigorous validation of phospho-specificity is essential for meaningful interpretation of results when using Phospho-ALOX5 (Ser523) antibodies. A comprehensive validation protocol includes:

• Lambda Phosphatase Treatment:

  • Divide sample into two equal portions

  • Treat one portion with lambda phosphatase (λ-PPase, 1200 units) for 30 minutes at 30°C

  • Run both treated and untreated samples on the same gel

  • Complete elimination of signal in the treated sample confirms phospho-specificity

• Phosphorylation Induction and Inhibition Controls:

  • Positive control: Treat cells with PKA activators (e.g., forskolin, cAMP analogs)

  • Negative control: Pre-treat cells with PKA inhibitors (e.g., H-89)

  • Compare signal intensity across conditions

• Peptide Competition Assay:

  • Pre-incubate antibody with phosphorylated peptide (containing Ser523)

  • Pre-incubate antibody with non-phosphorylated peptide (containing Ser523)

  • Compare immunoblotting results - specific signal should be blocked only by the phosphorylated peptide

• Site-Directed Mutagenesis:

  • Generate Ser523Ala mutant constructs

  • Express in appropriate cell systems

  • Compare antibody reactivity between wild-type and mutant samples

These validation methods ensure that observed signals genuinely represent ALOX5 phosphorylated at Ser523 rather than non-specific binding or cross-reactivity with other phosphorylation sites .

What signaling pathways regulate the PKA-mediated phosphorylation of ALOX5 at Ser523?

The PKA-mediated phosphorylation of ALOX5 at Ser523 is regulated by a complex network of signaling cascades:

• cAMP/PKA Canonical Pathway:

  • G-protein coupled receptor activation → adenylyl cyclase activation → increased cAMP → PKA activation → ALOX5 Ser523 phosphorylation

  • Receptor agonists: β-adrenergic receptor agonists, prostaglandin E2, adenosine

• Cross-talk with Other Pathways:

  • PKC pathway: Can indirectly modulate PKA activity

  • MAPK/ERK pathway: Phosphorylates ALOX5 at Ser663, potentially affecting Ser523 phosphorylation status

  • p38 MAPK/MK2 pathway: Phosphorylates ALOX5 at Ser271, with possible reciprocal regulation with Ser523 phosphorylation

• Cellular Stress Response Integration:

  • UV light, oxidative stress, osmotic shock, chemical stress, heat shock, and genotoxic agents activate p38 MK2 and ERK pathways

  • These stressors may indirectly affect Ser523 phosphorylation through pathway crosstalk

• Calcium Signaling Interaction:

  • Intracellular Ca2+ levels affect the relative importance of phosphorylation sites

  • The combination of phosphorylation at multiple sites enhances ALOX5 product synthesis when intracellular Ca2+ is low

Understanding these regulatory pathways provides opportunities for targeted modulation of ALOX5 activity in research and potential therapeutic applications.

How does ALOX5 Ser523 phosphorylation affect its interaction with other proteins in inflammatory pathways?

Phosphorylation at Ser523 significantly alters ALOX5's protein interaction network, directing inflammatory mediator production:

• ALOX5-cPLA2 Interaction:

  • When ALOX5 is not phosphorylated at Ser523, it preferentially interacts with cytosolic phospholipase A2 (cPLA2)

  • This interaction favors the production of pro-inflammatory leukotriene B4

  • The complex typically forms at the nuclear membrane

• ALOX5-COX2 Interaction:

  • PKA-mediated phosphorylation at Ser523 promotes interaction with cyclooxygenase-2 (COX2)

  • This interaction shifts production toward anti-inflammatory 15-epilipoxin-A4 (15-epi-LXA4)

  • May occur in different subcellular compartments compared to the ALOX5-cPLA2 complex

• ALOX5-FLAP Interaction:

  • Phosphorylation at Ser523 inhibits the association between ALOX5 and 5-lipoxygenase-activating protein (FLAP)

  • FLAP is required for efficient leukotriene synthesis

  • Reduced interaction impedes translocation of 5-lipoxygenase from the cytoplasm to the cell membrane

• Regulatory Protein Binding:

  • Phosphorylation creates binding sites for 14-3-3 proteins

  • These interactions may sequester ALOX5 in the cytoplasm, preventing nuclear translocation

  • May recruit additional regulatory proteins that modulate ALOX5 stability or activity

These interaction dynamics explain how phosphorylation status serves as a molecular switch directing inflammatory processes toward either pro-inflammatory or resolution pathways .

What experimental approaches can be used to study the interplay between ALOX5 phosphorylation and inflammatory disease models?

Researchers investigating ALOX5 phosphorylation in disease contexts can employ the following comprehensive experimental approaches:

• Animal Models with Pharmacological Interventions:

  • Administer PKA activators (e.g., forskolin) or inhibitors (e.g., H-89)

  • Use disease-specific models: ovalbumin-induced asthma, collagen-induced arthritis, atherosclerosis in ApoE-/- mice

  • Analyze tissue samples for ALOX5 phosphorylation status using Phospho-ALOX5 (Ser523) antibodies

  • Example approach: Rats receiving pioglitazone, atorvastatin, or combination therapy to study cardiac inflammation

• Site-Directed Mutagenesis and Transgenic Models:

  • Generate Ser523Ala (phospho-deficient) and Ser523Asp (phospho-mimetic) mutants

  • Create knock-in mice expressing these mutants

  • Compare inflammatory responses and disease progression between wild-type and mutant models

• Ex Vivo Tissue Analysis Protocol:

  • Collect tissue samples from patients with inflammatory conditions

  • Perform Western blotting with Phospho-ALOX5 (Ser523) antibodies

  • Correlate phosphorylation levels with disease severity and biomarkers

  • Conduct immunohistochemistry to assess tissue distribution of phosphorylated ALOX5

• Lipidomic Analysis of ALOX5 Metabolites:

  • Liquid chromatography-mass spectrometry (LC-MS/MS) profiling of leukotrienes and lipoxins

  • Correlate metabolite levels with ALOX5 phosphorylation status

  • Compare profiles between disease models and after treatment with pathway modulators

• Phosphorylation-Dependent Protein Interaction Studies:

  • Co-immunoprecipitation of ALOX5 with interaction partners (cPLA2, COX2)

  • Proximity ligation assays in disease-relevant tissues or cells

  • FRET/BRET assays to monitor interactions in real-time after stimulation

This integrative approach provides mechanistic insights into how ALOX5 phosphorylation status influences disease progression and response to therapies in various inflammatory conditions .

What are the optimal storage and handling conditions for Phospho-ALOX5 (Ser523) antibodies?

Proper storage and handling are critical for maintaining antibody functionality and experimental reproducibility:

The presence of 50% glycerol in the storage buffer allows researchers to take aliquots without complete freeze-thawing of the stock solution, which helps preserve antibody activity for longer periods . For Western blot applications, a working dilution of 1:1000 is typically recommended, though this may be optimized based on specific experimental conditions .

How can researchers resolve common issues with Phospho-ALOX5 (Ser523) antibody applications?

When working with Phospho-ALOX5 (Ser523) antibodies, researchers may encounter several technical challenges. The following troubleshooting guidelines address common issues:

• Weak or No Signal in Western Blot:

  • Verify phosphorylation status: Use positive controls with PKA activators

  • Prevent dephosphorylation: Include phosphatase inhibitors in all buffers

  • Optimize antibody concentration: Test dilutions from 1:500 to 1:2000

  • Enhance detection: Use high-sensitivity ECL substrates or increase exposure time

  • Enrich target protein: Consider immunoprecipitation before Western blotting

• High Background or Non-specific Bands:

  • Increase blocking stringency: Use 5% BSA instead of milk (phospho-epitopes)

  • Optimize washing: Extend TBST washing steps (at least 3 x 10 minutes)

  • Reduce antibody concentration: Try more dilute solutions

  • Pre-adsorb antibody: Incubate with non-phosphorylated peptide to remove non-specific antibodies

  • Use fresh transfer buffer: Avoid methanol evaporation which can affect transfer efficiency

• Inconsistent Results Between Experiments:

  • Standardize lysate preparation: Ensure consistent cell lysis conditions

  • Control phosphorylation status: Standardize cell stimulation protocols

  • Implement loading controls: Use total ALOX5 antibody on parallel blots

  • Prepare fresh working solutions: Avoid repeated freeze-thaw of diluted antibody

  • Establish consistent analysis parameters: Use densitometry with appropriate normalization

• Lambda Phosphatase Control Failures:

  • Verify enzyme activity: Use a known phosphoprotein as positive control

  • Optimize reaction conditions: Ensure proper buffer, temperature, and incubation time

  • Remove phosphatase inhibitors: Dialyze samples if necessary

  • Increase enzyme amount: Try up to 1600 units for difficult samples

  • Extend incubation time: Up to 60 minutes may be required for complete dephosphorylation

Implementing these technical solutions ensures more reliable and reproducible results when investigating ALOX5 phosphorylation in experimental systems.

What complementary techniques can enhance research findings when using Phospho-ALOX5 (Ser523) antibodies?

To maximize the scientific value of research utilizing Phospho-ALOX5 (Ser523) antibodies, investigators should consider implementing these complementary approaches:

• Parallel Phosphorylation Site Analysis:

  • Simultaneously detect multiple ALOX5 phosphorylation sites (Ser271, Ser663, Ser523)

  • Compare ratios of differently phosphorylated forms across experimental conditions

  • Correlate site-specific phosphorylation with functional outcomes

• Kinase Activity Assays:

  • Measure PKA activity directly using commercial kits

  • Correlate PKA activity with ALOX5 Ser523 phosphorylation levels

  • Assess changes in response to stimuli or inhibitors

• Real-time Translocation Monitoring:

  • Use fluorescently-tagged ALOX5 constructs (wild-type and Ser523 mutants)

  • Live-cell imaging to track subcellular localization

  • Quantify nuclear/cytoplasmic ratios in response to stimuli

• 3D Structural Analysis:

  • Molecular modeling of phosphorylated vs. non-phosphorylated ALOX5

  • Predict conformational changes affecting enzyme activity or protein interactions

  • Guide design of phosphorylation-specific inhibitors

• Quantitative Proteomic Analysis:

  • Phosphoproteomic profiling using mass spectrometry

  • Identify changes in the global phosphorylation landscape

  • Discover novel phosphorylation-dependent interaction partners

• Single-Cell Analysis:

  • Flow cytometry with phospho-specific antibodies

  • Assess cell-to-cell variability in ALOX5 phosphorylation

  • Correlate with cellular phenotypes or disease states

• CRISPR-Cas9 Genome Editing:

  • Generate cell lines with phospho-deficient ALOX5 (Ser523Ala)

  • Create phospho-mimetic mutations (Ser523Asp)

  • Compare inflammatory responses and leukotriene production

These complementary approaches provide a multi-dimensional perspective on ALOX5 regulation, strengthening the biological relevance and translational potential of findings obtained using Phospho-ALOX5 (Ser523) antibodies .

How does ALOX5 Ser523 phosphorylation influence inflammatory disease processes?

ALOX5 Ser523 phosphorylation serves as a critical regulatory mechanism in multiple inflammatory conditions:

• Asthma and Respiratory Inflammation:

  • Reduced Ser523 phosphorylation correlates with increased leukotriene production

  • Leukotrienes drive bronchoconstriction, mucus hypersecretion, and eosinophil recruitment

  • PKA activators can increase Ser523 phosphorylation, potentially limiting inflammation

• Cardiovascular Diseases:

  • Atorvastatin and pioglitazone upregulate 15-epi-LXA4 production in cardiac tissue

  • This effect may be mediated through changes in ALOX5 phosphorylation status

  • PKA inhibition with H-89 can block these beneficial effects, highlighting the importance of Ser523 phosphorylation

• Fibrotic Disorders:

  • Abnormal ALOX5 activity contributes to fibrosis in multiple organs

  • Phosphorylation at Ser523 may limit pro-fibrotic leukotriene production

  • Targeting this pathway represents a potential therapeutic strategy

• Neuroinflammation:

  • ALOX5 is expressed in brain tissue, particularly in microglia and neurons

  • Phosphorylated ALOX5 (Ser523) can be detected in rat hippocampal lysates

  • Modulation of this phosphorylation may impact neuroinflammatory processes

• Systemic Inflammatory Conditions:

  • ALOX5 activity contributes to arthritis pathogenesis

  • Ser523 phosphorylation status may determine whether pro-inflammatory or pro-resolving lipid mediators predominate

  • Therapeutic approaches targeting this phosphorylation site could modify disease progression

The ability to detect and quantify ALOX5 Ser523 phosphorylation using specific antibodies enables researchers to investigate these disease mechanisms and evaluate potential therapeutic interventions targeting this regulatory pathway .

What role does ALOX5 Ser523 phosphorylation play in the action of anti-inflammatory therapeutics?

The phosphorylation of ALOX5 at Ser523 represents a critical target for both existing and emerging anti-inflammatory therapies:

• Statins (HMG-CoA Reductase Inhibitors):

  • Atorvastatin has been shown to upregulate 15-epi-LXA4 production in cardiac tissue

  • This effect may involve PKA-mediated phosphorylation of ALOX5 at Ser523

  • Phosphorylation redirects ALOX5 activity from pro-inflammatory leukotriene production toward anti-inflammatory lipoxin synthesis

• Thiazolidinediones (PPARγ Agonists):

  • Pioglitazone affects ALOX5 pathway regulation

  • When combined with atorvastatin, creates synergistic effects on inflammatory mediator production

  • These effects are abolished by PKA inhibition with H-89, suggesting involvement of Ser523 phosphorylation

• cAMP-Elevating Agents:

  • β-adrenergic agonists, phosphodiesterase inhibitors, and adenylyl cyclase activators

  • Increase intracellular cAMP, activating PKA

  • Enhanced PKA activity promotes ALOX5 Ser523 phosphorylation

  • Reduced leukotriene synthesis contributes to anti-inflammatory effects

• ALOX5 Inhibitors:

  • Direct 5-lipoxygenase inhibitors (e.g., zileuton)

  • May be more effective in conditions with reduced ALOX5 Ser523 phosphorylation

  • Phosphorylation status could serve as a biomarker for predicting treatment response

• Emerging Targeted Approaches:

  • Peptide mimetics that stabilize the phosphorylated state of ALOX5

  • Small molecules that selectively bind phosphorylated ALOX5 and enhance its inhibition

  • PROTAC (proteolysis targeting chimeras) technology to selectively degrade non-phosphorylated, active ALOX5

Understanding how existing medications affect ALOX5 phosphorylation provides mechanistic insights and may guide the development of more targeted anti-inflammatory therapeutics with improved efficacy and reduced side effects .

What emerging technologies might advance our understanding of ALOX5 Ser523 phosphorylation?

Several cutting-edge technologies are poised to transform research on ALOX5 phosphorylation:

• Single-Cell Phosphoproteomics:

  • Enables analysis of ALOX5 phosphorylation heterogeneity within cell populations

  • Can reveal cell-specific responses to stimuli or inhibitors

  • Correlates phosphorylation status with cellular phenotypes at unprecedented resolution

• Phospho-Specific Nanobodies and Intrabodies:

  • Smaller than conventional antibodies with superior tissue penetration

  • Can be expressed intracellularly to track phosphorylation in live cells

  • Potential for therapeutic applications targeting specific phosphorylated forms

• CRISPR-Based Phosphorylation Reporters:

  • Direct genomic integration of fluorescent reporters linked to phosphorylation-dependent binding domains

  • Real-time monitoring of endogenous ALOX5 phosphorylation

  • Physiologically relevant readouts in intact biological systems

• Cryo-EM and Structural Biology:

  • High-resolution structures of ALOX5 in different phosphorylation states

  • Insights into conformational changes induced by Ser523 phosphorylation

  • Structure-based design of phosphorylation state-specific modulators

• Protein-Protein Interaction Networks:

  • Proximity labeling techniques (BioID, APEX) to identify phosphorylation-specific interactors

  • Spatial resolution of interaction dynamics using split enzyme complementation

  • Systems biology approaches to model phosphorylation-dependent signaling networks

• Organ-on-Chip and Advanced Disease Models:

  • Microphysiological systems recapitulating tissue-specific ALOX5 regulation

  • Patient-derived organoids to study phosphorylation in human disease contexts

  • Integration with biosensors for real-time monitoring of inflammatory mediators

These technological advances will provide unprecedented insights into how ALOX5 Ser523 phosphorylation regulates inflammatory processes at molecular, cellular, and tissue levels, potentially revealing new therapeutic opportunities .

What are the potential therapeutic implications of targeting ALOX5 Ser523 phosphorylation?

Targeting ALOX5 Ser523 phosphorylation represents a promising therapeutic strategy with several advantages over conventional approaches:

• Selective Modulation vs. Complete Inhibition:

  • Enhancing Ser523 phosphorylation selectively inhibits pro-inflammatory leukotriene production

  • Preserves or redirects ALOX5 activity toward anti-inflammatory mediator synthesis

  • May offer improved side effect profile compared to complete ALOX5 inhibition

• Precision Medicine Applications:

  • Phosphorylation status as biomarker for patient stratification

  • Personalized therapeutic approaches based on baseline ALOX5 phosphorylation

  • Monitoring phosphorylation as pharmacodynamic marker of treatment efficacy

• Novel Therapeutic Modalities:

  • Phosphorylation-stabilizing peptides mimicking the Ser523 region

  • Small molecule enhancers of PKA-mediated ALOX5 phosphorylation

  • Targeted protein degradation approaches specific for non-phosphorylated ALOX5

  • RNA therapeutics modulating expression of kinases/phosphatases regulating Ser523

• Combination Therapy Rationales:

  • Synergistic effects of phosphorylation enhancers with existing anti-inflammatory agents

  • Dual targeting of different phosphorylation sites to comprehensively modulate ALOX5 function

  • Combination with drugs affecting downstream leukotriene receptors

• Disease-Specific Applications:

  • Asthma: Enhancing Ser523 phosphorylation to complement bronchodilator therapy

  • Cardiovascular disease: Promoting anti-inflammatory lipoxin production through ALOX5 phosphorylation

  • Neuroinflammation: Brain-penetrant modulators of ALOX5 phosphorylation

  • Chronic inflammatory conditions: Long-term modulation as maintenance therapy