Phospho-NFE2L2 (S40) Recombinant Monoclonal Antibody

What is the biological significance of NFE2L2 phosphorylation at serine 40?

Phosphorylation of NFE2L2 (also known as Nrf2) at serine 40 (S40) represents a critical regulatory mechanism in the cellular oxidative stress response pathway. This specific post-translational modification disrupts the interaction between NFE2L2 and KEAP1, preventing ubiquitination and subsequent proteasomal degradation of NFE2L2. Consequently, phosphorylated NFE2L2 accumulates in the nucleus where it heterodimerizes with small Maf proteins and binds to antioxidant response elements (ARE) in the promoter regions of cytoprotective genes. This activation triggers the expression of phase 2 detoxifying enzymes and antioxidant proteins that neutralize reactive electrophiles and protect cells from oxidative damage . The S40 phosphorylation site is particularly important as a direct target of protein kinase C (PKC) during oxidative stress conditions, making it a key biomarker for monitoring NFE2L2 activation status in various experimental contexts.

How does the NFE2L2 pathway interact with cellular stress response mechanisms?

The NFE2L2 pathway demonstrates remarkable integration with multiple cellular stress response mechanisms:

• Oxidative Stress Response: NFE2L2 acts as the master regulator of antioxidant defenses by upregulating genes that neutralize reactive oxygen species and electrophiles .

• Selective Autophagy: The NFE2L2 pathway is activated during selective autophagy, where autophagy promotes interaction between KEAP1 and SQSTM1/p62, leading to inactivation of the BCR(KEAP1) complex and subsequent NFE2L2 nuclear accumulation .

• Unfolded Protein Response (UPR): During endoplasmic reticulum stress, the NFE2L2 pathway is activated to maintain redox homeostasis and promote cell survival .

• Inflammatory Response: NFE2L2 suppresses macrophage inflammatory responses by blocking pro-inflammatory signaling pathways, serving as a critical regulator of innate immune responses and survival during sepsis .

• Ferroptosis Regulation: NFE2L2 activation can inhibit ferroptosis, a form of regulated cell death characterized by iron-dependent lipid peroxidation, by upregulating various target genes involved in iron metabolism and antioxidant defense .

This multifaceted involvement makes phospho-NFE2L2 detection crucial for studying integrated stress responses across various experimental models.

What are the optimal applications for Phospho-NFE2L2 (S40) Recombinant Monoclonal Antibody in research?

Phospho-NFE2L2 (S40) Recombinant Monoclonal Antibody demonstrates validated utility across multiple experimental techniques:

• Western Blot (WB): Provides quantitative analysis of phospho-NFE2L2 levels in cell and tissue lysates, allowing assessment of activation status across different experimental conditions .

• Immunohistochemistry (IHC): Enables visualization of phospho-NFE2L2 localization within tissue sections, particularly useful for assessing nuclear translocation as an indicator of activation .

• Immunocytochemistry/Immunofluorescence (ICC/IF): Allows high-resolution subcellular localization studies to track phospho-NFE2L2 translocation between cytoplasm and nucleus in response to various stimuli .

• Flow Cytometry: Permits quantitative single-cell analysis of phospho-NFE2L2 levels within heterogeneous cell populations, enabling identification of responsive subpopulations .

• Chromatin Immunoprecipitation (ChIP): While not explicitly mentioned in the search results, this technique can be employed to assess phospho-NFE2L2 binding to ARE sequences in target gene promoters.

The choice of application should be guided by specific research questions, with western blot and immunofluorescence being particularly valuable for monitoring activation dynamics in response to oxidative challenges.

How should samples be prepared to maximize phospho-NFE2L2 (S40) detection?

Optimal sample preparation for phospho-NFE2L2 (S40) detection requires careful consideration of the following methodological aspects:

• Lysis Buffer Composition: Use phosphatase inhibitor-enriched lysis buffers (containing sodium fluoride, sodium orthovanadate, and β-glycerophosphate) to preserve phosphorylation status during extraction.

• Nuclear Fraction Isolation: Since activated phospho-NFE2L2 translocates to the nucleus, nuclear extraction protocols yield enriched samples for detection of the activated form. Standard protocols using hypotonic lysis followed by high-salt nuclear extraction are recommended.

• Rapid Processing: Process samples quickly at 4°C to minimize dephosphorylation by endogenous phosphatases.

• Fixation for Microscopy: For immunofluorescence detection, paraformaldehyde fixation (4%) followed by permeabilization with 0.1% Triton X-100 preserves phospho-epitopes while enabling antibody access.

• Positive Controls: Include samples treated with known NFE2L2 activators (such as sulforaphane or tert-butylhydroquinone) as positive controls to validate antibody performance.

Additionally, when performing western blot analysis, use freshly prepared samples whenever possible, as freeze-thaw cycles can diminish phospho-epitope integrity and affect detection sensitivity.

How can researchers overcome common challenges in phospho-NFE2L2 (S40) detection?

Researchers frequently encounter several technical challenges when detecting phospho-NFE2L2 (S40), which can be addressed through the following approaches:

• High Background Signal:

  • Increase blocking time (5% BSA in TBST for 2 hours)

  • Optimize primary antibody dilution (typically 1:500-1:2000)

  • Include additional washing steps (5 x 5 minutes in TBST)

  • Use more specific secondary antibodies with minimal cross-reactivity

• Weak or Absent Signal:

  • Confirm NFE2L2 activation with positive controls (e.g., H₂O₂ treatment)

  • Enrich nuclear fractions where activated phospho-NFE2L2 accumulates

  • Optimize protein loading (50-100 μg for whole cell lysates)

  • Increase exposure time for chemiluminescence detection

• Multiple Bands in Western Blot:

  • Use gradient gels (4-12%) to better resolve NFE2L2 isoforms

  • Include protease inhibitors in lysis buffer to prevent degradation

  • Validate bands with positive controls and knockout/knockdown samples

• Poor Nuclear Staining in Immunofluorescence:

  • Optimize permeabilization conditions (0.1-0.5% Triton X-100)

  • Use antigen retrieval for fixed tissues (citrate buffer pH 6.0)

  • Include DAPI nuclear counterstain for proper localization assessment

These methodological refinements significantly improve detection reliability while minimizing artifacts that could lead to misinterpretation of experimental results.

What are the critical controls needed when working with Phospho-NFE2L2 (S40) antibodies?

Rigorous experimental design requires incorporation of several key controls to ensure valid interpretation of phospho-NFE2L2 (S40) detection:

• Positive Controls:

  • Cells/tissues treated with known NFE2L2 activators (sulforaphane, tert-butylhydroquinone)

  • Recombinant phosphorylated NFE2L2 protein (for western blot standard curves)

• Negative Controls:

  • Genetic models: NFE2L2 knockout/knockdown samples

  • Dephosphorylation controls: Lysate aliquots treated with lambda phosphatase

  • Secondary antibody-only controls to assess non-specific binding

• Specificity Controls:

  • Peptide competition assays using phospho-S40 peptides

  • Parallel detection with total NFE2L2 antibody to calculate phosphorylation ratio

  • Cross-validation with alternative phospho-NFE2L2 antibody clones

• Functional Validation:

  • ARE-luciferase reporter assays to confirm transcriptional activity

  • qPCR analysis of NFE2L2 target genes (NQO1, HMOX1, GCLM)

  • Subcellular fractionation to confirm nuclear translocation

Implementation of these controls provides crucial evidence for antibody specificity and ensures that observed signals genuinely represent phosphorylated NFE2L2 rather than artifacts or non-specific binding.

How can Phospho-NFE2L2 (S40) antibodies be utilized to study NFE2L2's role in ferroptosis resistance?

The application of phospho-NFE2L2 (S40) antibodies offers sophisticated approaches to investigate NFE2L2's critical role in ferroptosis resistance:

• Mechanistic Analysis: These antibodies enable direct monitoring of NFE2L2 activation status in response to ferroptosis inducers (e.g., erastin, RSL3), providing temporal resolution of the antioxidant response. This approach reveals whether NFE2L2 activation precedes, coincides with, or follows lipid peroxidation events, helping establish causality in protection mechanisms .

• Target Gene Expression Correlation: By combining phospho-NFE2L2 detection with analysis of downstream targets specifically involved in ferroptosis protection (SLC7A11, GPX4, FTH1, FTL), researchers can establish direct functional relationships between NFE2L2 activation and the expression of ferroptosis defense genes .

• Cancer Therapy Resistance Models: In cancer cell lines and patient-derived xenografts showing therapy resistance, phospho-NFE2L2 antibodies can identify whether constitutive activation contributes to ferroptosis evasion. This information guides rational design of combination therapies targeting both NFE2L2 and ferroptosis pathways .

• Structure-Function Analysis: When combined with site-directed mutagenesis of NFE2L2 domains, these antibodies help determine how S40 phosphorylation specifically contributes to ferroptosis resistance compared to other activation mechanisms.

• Drug Discovery Applications: The antibodies facilitate high-throughput screening of compounds that modulate NFE2L2 phosphorylation status, potentially identifying novel ferroptosis sensitizers for cancer therapy .

A methodical research approach using these techniques has revealed that NFE2L2 activation upregulates multiple ferroptosis defense genes including SLC7A11, GCL, GSS, GSR, GPX4, and metabolic enzymes that support glutathione synthesis and utilization .

What methodological approaches can differentiate between KEAP1-dependent and KEAP1-independent NFE2L2 activation?

Distinguishing between KEAP1-dependent and KEAP1-independent NFE2L2 activation requires sophisticated methodological approaches:

• Phosphorylation Status Analysis: Phospho-NFE2L2 (S40) antibodies are particularly valuable as S40 phosphorylation represents a KEAP1-independent activation mechanism. Comparative analysis of total NFE2L2 versus phospho-S40 NFE2L2 levels can establish the relative contribution of each pathway:

  Activation Mechanism	Total NFE2L2	Phospho-S40 NFE2L2	Nuclear/Cytoplasmic Ratio
  KEAP1-dependent	Increased	Minimal change	Increased
  Phosphorylation-dependent	Moderate increase	Significantly increased	Increased
  Combined mechanisms	Substantially increased	Significantly increased	Substantially increased

• Protein-Protein Interaction Analysis:

  • Co-immunoprecipitation assays using phospho-NFE2L2 (S40) antibodies to assess interaction with KEAP1

  • Proximity ligation assays to visualize NFE2L2-KEAP1 interactions in situ

  • FRET-based biosensors to monitor real-time dissociation kinetics

• Genetic Approaches:

  • KEAP1 knockout or knockdown studies combined with phospho-NFE2L2 (S40) detection

  • Expression of phospho-mimetic (S40D) or phospho-deficient (S40A) NFE2L2 mutants

  • CRISPR-Cas9 genome editing of endogenous NFE2L2 phosphorylation sites

• Pathway-Specific Activators:

  • Electrophiles like sulforaphane (predominantly KEAP1-dependent)

  • PKC activators like PMA (predominantly phosphorylation-dependent)

  • Autophagy modulators affecting SQSTM1/p62-mediated KEAP1 sequestration

These approaches revealed that multiple regulatory mechanisms often operate simultaneously, with phosphorylation at S40 facilitating the escape from KEAP1-mediated degradation while also independently enhancing NFE2L2 transcriptional activity through altered protein-protein interactions with transcriptional machinery .

How can researchers investigate the interplay between NFE2L2 phosphorylation and its role in regulating innate immune responses?

Investigating the complex relationship between NFE2L2 phosphorylation and innate immune regulation requires multidisciplinary experimental approaches:

• Temporal Activation Studies:

  • Use phospho-NFE2L2 (S40) antibodies to track activation kinetics during immune challenges

  • Perform parallel analysis of NFE2L2 phosphorylation and inflammatory cytokine production

  • Establish time-dependent relationships between NFE2L2 activation and resolution phases of inflammation

• Cell Type-Specific Analysis:

  • Flow cytometry with phospho-NFE2L2 (S40) antibodies to identify responsive immune cell populations

  • Single-cell resolution of NFE2L2 activation in heterogeneous immune cell populations

  • Correlation of phospho-NFE2L2 levels with immune cell functional states

• Signaling Pathway Cross-talk:

  • Investigate relationships between TLR/MyD88 signaling and NFE2L2 phosphorylation

  • Assess how TNF-alpha signaling influences NFE2L2 phosphorylation status

  • Examine reciprocal regulation between NF-κB and phospho-NFE2L2 pathways

• Functional Outcome Assessment:

  • Measure how manipulation of NFE2L2 phosphorylation affects phagocytosis, ROS production, and cytokine secretion

  • Correlate phospho-NFE2L2 levels with bacterial clearance or viral replication in infection models

  • Assess the impact on resolution of inflammation and tissue damage

• Disease Model Applications:

  • Study phospho-NFE2L2 dynamics in sepsis models to understand its protective role

  • Investigate activation patterns in chronic inflammatory conditions

  • Examine how pharmacological modulation of NFE2L2 phosphorylation affects disease outcomes

Research using these approaches has demonstrated that NFE2L2 acts as a critical regulator of innate immune responses during sepsis by maintaining redox homeostasis and restraining dysregulated pro-inflammatory signaling pathways, including MyD88-dependent and -independent pathways and TNF-alpha signaling . The phosphorylation status of NFE2L2 serves as a molecular switch that balances protective inflammatory responses with excessive immune activation.

How can Phospho-NFE2L2 (S40) antibodies be utilized in studying therapeutic resistance mechanisms in cancer?

Phospho-NFE2L2 (S40) antibodies provide powerful tools for elucidating the mechanisms of therapeutic resistance in cancer:

• Biomarker Development:

  • Quantitative analysis of phospho-NFE2L2 (S40) levels in patient tumor samples to predict therapy response

  • Correlation of NFE2L2 phosphorylation status with clinical outcomes and resistance patterns

  • Longitudinal monitoring of phospho-NFE2L2 levels during treatment to detect emerging resistance

• Resistance Mechanism Characterization:

  • Comparative phospho-NFE2L2 profiling between sensitive and resistant cell lines

  • Analysis of NFE2L2 activation following exposure to chemotherapeutics, radiation, or targeted therapies

  • Identification of resistance-associated NFE2L2 target gene signatures

• Combination Therapy Development:

  • Screening for compounds that specifically inhibit NFE2L2 phosphorylation at S40

  • Testing combinations of phospho-NFE2L2 inhibitors with ferroptosis inducers

  • Evaluation of sequential therapy approaches targeting NFE2L2-dependent resistance mechanisms

• Mechanistic Studies:

  • Investigation of how tumor microenvironment factors regulate NFE2L2 phosphorylation

  • Analysis of cross-talk between NFE2L2 and other resistance-associated signaling pathways

  • Identification of kinases responsible for therapy-induced NFE2L2 phosphorylation

Research has demonstrated that elevated NFE2L2 activity supports tumor progression, metastasis, and therapy resistance in various malignancies. NFE2L2 activation contributes to drug resistance by upregulating antioxidant defenses and inhibiting ferroptosis through regulation of multiple target genes including NQO1, HMOX1, FTH1, FTL, SLC7A11, GCL, GSS, GSR, GPX4, and AIFM2 . Phospho-NFE2L2 (S40) antibodies enable precise tracking of this activation, making them invaluable tools for developing strategies to overcome NFE2L2-mediated therapeutic resistance.

What methodological approaches can resolve contradictory data regarding NFE2L2 activation in different experimental models?

Resolving contradictory data regarding NFE2L2 activation across different experimental models requires systematic methodological approaches:

• Standardized Detection Protocols:

  • Implement consistent antibody validation procedures across laboratories

  • Establish standardized positive and negative controls for phospho-NFE2L2 detection

  • Develop quantitative assays with defined dynamic ranges and detection limits

• Comprehensive Activation Assessment:

  • Measure multiple NFE2L2 activation parameters simultaneously:
    a) Total protein levels (stability)
    b) Phosphorylation status (S40 and other sites)
    c) Nuclear translocation (subcellular fractionation)
    d) DNA binding activity (ChIP or EMSA)
    e) Transcriptional output (target gene expression)

• Context-Dependent Analysis:

  • Systematically evaluate how experimental variables affect NFE2L2 activation:
    a) Cell type and differentiation state
    b) Culture conditions (2D vs. 3D, oxygen tension)
    c) Acute vs. chronic stress exposure
    d) Presence of competing signaling pathways

• Time-Course Resolution:

  • Perform high-temporal resolution studies to capture activation kinetics

  • Distinguish between transient vs. sustained activation patterns

  • Correlate phosphorylation dynamics with functional outcomes

• Multi-Omics Integration:

  • Combine phospho-proteomics, transcriptomics, and metabolomics data

  • Develop computational models that predict context-dependent NFE2L2 responses

  • Identify factors that modify NFE2L2 activity in different experimental systems

This methodical approach has revealed that apparent contradictions often reflect genuine biological complexity rather than technical artifacts. For example, in certain contexts, NFE2L2 phosphorylation may be disconnected from nuclear translocation due to additional regulatory mechanisms. Similarly, the transcriptional output of phospho-NFE2L2 can vary dramatically depending on the availability of co-factors and the epigenetic landscape of target genes .

What experimental design provides the most robust assessment of NFE2L2 activation status?

A comprehensive experimental design to robustly assess NFE2L2 activation status should incorporate multiple complementary approaches:

• Multi-parameter Assessment Strategy:

  Parameter	Technique	Information Provided
  Phosphorylation (S40)	Western blot with phospho-specific antibody	Direct measure of activation-associated modification
  Nuclear Translocation	Nuclear/cytoplasmic fractionation followed by western blot or immunofluorescence	Subcellular localization indicating activation
  KEAP1 Interaction	Co-immunoprecipitation or proximity ligation assay	Release from inhibitory complex
  DNA Binding	Chromatin immunoprecipitation (ChIP) or electrophoretic mobility shift assay (EMSA)	Functional engagement with target genes
  Transcriptional Activity	qPCR of target genes or ARE-luciferase reporter assay	Functional output of activation

• Temporal Resolution:

  • Early activation markers: S40 phosphorylation (minutes to hours)

  • Intermediate markers: Nuclear accumulation (hours)

  • Late markers: Target gene expression (hours to days)

  • Include multiple time points to capture the complete activation cycle

• Stimulus-Specific Responses:

  • Compare phospho-NFE2L2 responses across different activators:
    a) Electrophiles (sulforaphane, tBHQ)
    b) Oxidative stressors (H₂O₂, paraquat)
    c) Pathway-specific activators (PKC activators for phosphorylation)

  • Determine stimulus-specific activation signatures

• Genetic Validation:

  • Include CRISPR-engineered cell lines with phospho-site mutations (S40A)

  • Use NFE2L2 or KEAP1 knockout models as controls

  • Employ reconstitution experiments with wild-type vs. mutant NFE2L2

This integrated approach provides a comprehensive assessment of NFE2L2 activation status while controlling for technical artifacts and biological variability. Studies employing such robust designs have revealed that NFE2L2 activation involves a coordinated sequence of events, with phosphorylation at S40 often serving as an early indicator that precedes and facilitates subsequent steps in the activation cascade .

How should researchers interpret conflicting results between phospho-NFE2L2 levels and downstream target gene expression?

Interpreting discrepancies between phospho-NFE2L2 (S40) levels and target gene expression requires systematic analysis of potential mechanistic explanations:

• Temporal Disconnection:

  • Phosphorylation typically precedes transcriptional changes by several hours

  • Establish time-course relationships before concluding genuine disconnection

  • Consider target gene mRNA half-life and protein turnover rates when interpreting expression data

• Threshold Effects:

  • Determine whether minimum threshold levels of phospho-NFE2L2 are required for transcriptional activation

  • Establish dose-response relationships between phosphorylation levels and target gene expression

  • Consider that different target genes may have different activation thresholds

• Competing Regulatory Mechanisms:

  • Investigate epigenetic regulation of target gene promoters (histone modifications, DNA methylation)

  • Assess availability of essential co-factors (small Maf proteins, co-activators)

  • Examine presence of competing transcription factors at ARE sites

  • Consider post-transcriptional regulation of target mRNAs

• Technical Considerations:

  • Verify antibody specificity using appropriate controls

  • Ensure nuclear extraction protocols effectively isolate transcriptionally active phospho-NFE2L2

  • Validate target gene primer specificity and efficiency

• Integrated Analysis Approach:

  • Implement single-cell analyses to resolve population heterogeneity

  • Use ChIP-seq to map genome-wide NFE2L2 binding in relation to phosphorylation status

  • Apply mathematical modeling to integrate multiple regulatory layers

Research utilizing these analytical approaches has revealed that while S40 phosphorylation enhances NFE2L2 transcriptional activity, it is not always sufficient for target gene induction. For example, studies examining NFE2L2 binding to ARE sequences in chagasic myocardium demonstrated that despite the presence of phospho-NFE2L2, binding capacity was decreased by 52%, leading to reduced expression of downstream targets like MnSOD, γGCS, and HO1 . This indicates that additional factors beyond phosphorylation status regulate the functional output of the NFE2L2 pathway.