NFE2L3 Antibody

What is NFE2L3 and why is it important in research?

NFE2L3 is a 694-amino acid transcription factor belonging to the Cap'n'Collar (CNC) family. It plays crucial roles in transcriptional regulation via RNA polymerase II and is involved in multiple biological processes including carcinogenesis, stress response, differentiation, and inflammatory processes. NFE2L3 has emerged as a stemness marker gene due to its early upregulation during stem cell differentiation . Recent studies have implicated NFE2L3 in cancer biology, particularly in colorectal cancer and malignant pleural mesothelioma, making it an important research target .

How do I select the appropriate NFE2L3 antibody for my experiment?

Selection should be based on:

• Experimental application: Verify validation for your specific application (WB, IHC, IF, ELISA)

• Species reactivity: Ensure reactivity with your model organism (human, mouse, rat)

• Isoform recognition: Consider whether all three forms (A, B, C) need to be detected

• Epitope location: Choose antibodies targeting different regions based on your research question

• Validation data: Review published literature and supplier validation images

Note that NFE2L3 is highly expressed in human placenta and B-cell/monocyte cell lines, making these useful positive controls .

How should I store and handle NFE2L3 antibodies for optimal performance?

NFE2L3 antibodies typically require storage at -20°C for long-term stability. For frequent use within a month, 4°C storage is acceptable. Avoid repeated freeze-thaw cycles as they can degrade antibody performance. Most NFE2L3 antibodies are supplied in PBS containing preservatives like 50% glycerol, 0.5% BSA, and 0.02% sodium azide . When diluting for experiments, always use fresh buffer systems and consider adding protein carriers (BSA, non-fat milk) to prevent non-specific binding and improve signal-to-noise ratio.

How can I detect the three different forms of NFE2L3 protein (A, B, C) in cellular fractions?

Detection of the three forms requires careful subcellular fractionation and antibody selection:

• Sample preparation: Utilize differential centrifugation or commercial fractionation kits (e.g., Minute™ Cytoplasmic and Nuclear Fractionation kit)

• Loading controls: Include compartment-specific markers (e.g., GAPDH for cytoplasm, Lamin B for nucleus, Calnexin for ER)

• Gel conditions: Use 8-10% polyacrylamide gels with extended run times to resolve the three forms

• Form identification: Form A (~76 kDa) associates with ER, form B is predominantly cytoplasmic, and form C localizes to the nucleus

Western blot analysis of specific cellular compartments is critical as combining all fractions may obscure the different forms. For validation, immunofluorescence microscopy with co-staining of compartment markers can confirm subcellular localization .

What controls should I include when studying NFE2L3 expression and localization?

Essential controls include:

• Positive tissue control: Human placenta tissue expresses high levels of NFE2L3

• Negative controls: Primary antibody omission and isotype controls

• Knockdown/knockout validation: siRNA or CRISPR-mediated NFE2L3 depletion

• Proteasome inhibition: MG-132 treatment to prevent degradation and enhance detection

• Blocking peptide controls: To confirm specificity, especially in immunostaining applications

For localization studies, include specific organelle markers (nuclear, ER, cytoplasmic) to confirm the differential distribution of the three NFE2L3 forms .

How should I optimize NFE2L3 detection in Western blot given its short half-life?

NFE2L3 has a remarkably short half-life (20-40 minutes), making detection challenging . Optimization strategies include:

• Proteasome inhibition: Treat cells with MG-132 or other proteasomal inhibitors (β-lactacystin, epoxomicin) 2-4 hours before harvest

• Protein extraction buffer: Include phosphatase inhibitors, deubiquitinase inhibitors, and strong detergents

• GSK3 inhibition: Use lithium chloride to inhibit GSK3, which is involved in NFE2L3 degradation

• Rapid sample processing: Minimize time between cell lysis and denaturation

• Sample denaturation: Use LDS sample buffer with higher SDS concentration than standard Laemmli buffer

The above approaches can significantly improve the detection of all three forms, with the B and C forms showing greater stabilization upon proteasome inhibition .

How can I study NFE2L3 post-translational modifications and degradation pathways?

NFE2L3 undergoes polyubiquitination and proteasomal degradation. To study these processes:

• Ubiquitination assays: Co-transfect cells with NFE2L3 and HA-tagged ubiquitin constructs, then perform immunoprecipitation with NFE2L3 antibodies followed by immunoblotting for HA

• Half-life determination: Perform cycloheximide chase assays with and without proteasome inhibitors

• GSK3-mediated regulation: Use GSK3 inhibitors (lithium chloride) or shRNA-mediated knockdown to assess impact on NFE2L3 stability

• Phosphorylation assessment: Use phospho-specific antibodies or PhosTag gels to detect phosphorylated forms

• PEST motif analysis: Create deletion constructs to evaluate the contribution of the PEST motif to protein stability

A comprehensive approach should include both gain-of-function studies with overexpression systems and loss-of-function approaches with targeted knockdown/knockout methods.

What experimental approaches can determine NFE2L3's role in cancer and inflammation?

Based on recent literature, NFE2L3 plays complex roles in cancer and inflammation. Investigation strategies include:

• Genetic models: Utilize Nfe2l3-/- knockout mice in cancer and inflammation models

• Tumor microenvironment analysis: Perform digital spatial profiling and immunohistochemistry for immune cell markers

• Cytokine profiling: Measure IL-33 and other cytokines regulated by NFE2L3

• ChIP experiments: Conduct chromatin immunoprecipitation to identify direct transcriptional targets

• RNA-seq and pathway analysis: Assess transcriptome changes upon NFE2L3 modulation

These approaches have revealed that NFE2L3 loss reduces inflammation in colorectal cancer models, affects mast cell abundance, and influences regulatory T cell populations in the tumor microenvironment .

How can NFE2L3 antibodies be used to study its interaction with the IL-2/STAT5/NLRP3 pathway?

Recent research has identified connections between NFE2L3 and the IL-2/STAT5/NLRP3 pathway in malignancies like mesothelioma . Experimental approaches include:

• Co-immunoprecipitation: Use NFE2L3 antibodies to pull down complexes and blot for pathway components

• Proximity ligation assays: Detect protein-protein interactions between NFE2L3 and pathway components

• ChIP-seq analysis: Identify genomic binding regions of NFE2L3 near IL-2R, STAT5, or NLRP3 genes

• Transcriptional reporter assays: Measure impact of NFE2L3 on promoter activity of pathway genes

• Correlation analysis: Quantify expression correlations between NFE2L3 and IL-2RA, IL-2RB, IL-2RG, STAT5B, and NLRP3

These correlations suggest functional relationships that can be experimentally validated .

Why might I observe different banding patterns when using different NFE2L3 antibodies?

Variable banding patterns may result from:

• Different epitopes: Antibodies targeting different regions may recognize specific forms preferentially

• Post-translational modifications: Phosphorylation, ubiquitination, or proteolytic cleavage can alter migration

• Sample preparation: Nuclear vs. cytoplasmic vs. whole-cell extraction affects form distribution

• Protein degradation: The short half-life of NFE2L3 leads to degradation products

• Cross-reactivity: Some antibodies may detect related CNC family members

To resolve these issues, use antibodies raised against different epitopes, perform immunodepletion with competing antigens, and validate with siRNA knockdown controls .

How should I interpret NFE2L3 expression data in the context of cancer research?

Interpretation should consider:

• Subcellular localization: Nuclear vs. cytoplasmic expression may have different functional implications

• Cancer type specificity: NFE2L3 shows opposite roles in different cancers (e.g., oncogenic in esophageal cancer but suppressive in TNBC)

• Correlation with clinical outcomes: Higher expression associates with poor prognosis in some cancers

• Relation to inflammation markers: NFE2L3 correlates with inflammatory signatures in colorectal and mesothelioma cases

• Epigenetic regulation: DNA methylation status of NFE2L3 may influence expression and function

Research has shown that NFE2L3 can function as either an oncogene or tumor suppressor depending on the cellular context, requiring careful interpretation within specific cancer types .

What strategies can resolve contradictory results when studying NFE2L3 function across different cell types?

Researchers frequently encounter contradictory findings when studying NFE2L3 across different cellular systems. Resolution strategies include:

• Cell-type specific analysis: Compare NFE2L3 interactome in different cell types using IP-MS approaches

• Context-dependent signaling: Investigate upstream regulators and downstream effectors in each cell type

• Isoform-specific functions: Determine which NFE2L3 forms predominate in different cells

• Combinatorial approaches: Use both gain- and loss-of-function studies in parallel

• Single-cell analysis: Examine heterogeneity of NFE2L3 expression and function at single-cell resolution

These approaches can help reconcile seemingly contradictory results and reveal cell-type specific functions of NFE2L3 in different biological contexts.

How can NFE2L3 antibodies be used for biomarker development in clinical settings?

NFE2L3 shows promise as a biomarker in several cancers. Development approaches include:

• Tissue microarray analysis: Screen large cohorts of patient samples for NFE2L3 expression

• Correlation with clinical parameters: Associate expression with survival, treatment response, and disease stage

• Multiplex immunohistochemistry: Combine NFE2L3 staining with immune cell markers

• Liquid biopsy applications: Detect NFE2L3 or its mutations in circulating tumor DNA

• Methylation analysis: Assess NFE2L3 promoter methylation status as a prognostic indicator

Some cancers show that NFE2L3 hypermethylation correlates with better prognosis, suggesting epigenetic regulation as a potential biomarker approach .

What novel methodologies could enhance the study of NFE2L3's role in transcriptional regulation?

Cutting-edge approaches include:

• CUT&RUN or CUT&Tag: Map NFE2L3 binding sites with higher resolution than traditional ChIP

• HiChIP analysis: Identify long-range chromatin interactions mediated by NFE2L3

• Single-molecule imaging: Track NFE2L3 dynamics in living cells using fluorescent tags

• CRISPR activation/repression systems: Manipulate NFE2L3 expression at endogenous loci

• Proteomics approaches: Identify NFE2L3 interactors under different cellular conditions

These methodologies can provide deeper insights into NFE2L3's transcriptional functions beyond traditional approaches .

How might NFE2L3 research connect to emerging therapeutic strategies in cancer and inflammatory diseases?

Therapeutic implications include:

• Targeted protein degradation: Design PROTACs (proteolysis targeting chimeras) to modulate NFE2L3 levels

• Immune checkpoint modulation: Exploit NFE2L3's connection to FOXP3+ Tregs and immune checkpoints (CTLA4, TIM3, LAG3)

• Small molecule inhibitors: Develop compounds targeting the NFE2L3 pathway

• Combination therapies: Target NFE2L3 alongside IL-33/mast cell pathways

• Biomarker-guided therapy: Use NFE2L3 expression or methylation status to stratify patients for treatment

Research in colorectal cancer models demonstrates that NFE2L3 loss affects tumor sidedness and immune cell composition, suggesting potential for developing location-specific therapeutic strategies for early-onset colorectal cancer .