nfil3 Antibody

What is NFIL3 and what structural features should researchers consider when selecting antibodies?

NFIL3, also known as E4BP4, IL3BP1, NF-IL3A, or NFIL3A, is a transcription factor belonging to the bZIP family and NFIL3 subfamily. The protein has a molecular weight of approximately 51.5 kilodaltons and functions as a transcriptional repressor . When selecting antibodies, researchers should consider:

• Epitope location: Antibodies targeting different regions of NFIL3 may have varying specificities

• Cross-reactivity: Many commercial antibodies recognize multiple species orthologs including human, mouse, rat, and others

• Application compatibility: Different antibody clones may perform optimally in specific applications

The protein's structural domains include a DNA-binding domain and regulatory regions that interact with other transcription factors, which may influence antibody accessibility in certain applications.

What are the validated applications for NFIL3 antibodies?

NFIL3 antibodies have been validated for multiple experimental applications with specific recommended dilution ratios:

These applications enable researchers to detect NFIL3 in various experimental contexts, from protein expression analysis to localization studies . Optimization for each specific experimental system is strongly recommended.

How does NFIL3 expression vary across immune cell subsets?

NFIL3 exhibits differential expression patterns across immune cell populations:

• Regulatory T cells (Tregs) show lower NFIL3 expression compared to other CD4+ T cell subsets

• Naïve CD8+ T cells have nearly undetectable NFIL3 levels, but expression increases significantly during activation and differentiation into cytotoxic T lymphocytes (CTLs)

• NK cells, dendritic cells, and various CD4+ T cell subsets express NFIL3 with distinct temporal patterns

When designing experiments to detect NFIL3 in specific immune populations, researchers should consider these baseline expression differences and select appropriate positive and negative controls accordingly.

What are the optimal methods for detecting nuclear localization of NFIL3?

NFIL3 functions as a transcription factor with predominant nuclear localization. Research has demonstrated that in activated CD8+ T cells, NFIL3 is constitutively localized in the nucleus . For optimal detection:

• Cell fractionation approach:

  • Use nuclear extraction buffers with protease inhibitors

  • Confirm fraction purity with nuclear (e.g., Lamin B) and cytoplasmic (e.g., GAPDH) markers

  • Western blot analysis shows NFIL3 is almost exclusively present in nuclear fractions of activated T cells

• Immunofluorescence method:

  • Fix cells with 4% paraformaldehyde

  • Permeabilize with 0.1-0.5% Triton X-100

  • Use recommended antibody dilutions (1:400-1:1600)

  • Include nuclear counterstain (DAPI or Hoechst)

  • Confocal microscopy confirms nuclear localization pattern

These approaches have successfully demonstrated that NFIL3 is predominantly nuclear in differentiated CTLs, irrespective of differentiation status .

How can I design effective CRISPR-based experiments to study NFIL3 function?

CRISPR-Cas9 technology has been effectively used to elucidate NFIL3 function in CTLs. When designing similar experiments:

• Guide RNA design considerations:

  • Target conserved functional domains of NFIL3

  • Avoid regions with known single nucleotide polymorphisms

  • Design multiple guides to ensure efficiency and validate with sequencing

• Delivery method optimization:

  • For primary T cells, ribonucleoprotein (RNP) transfection has proven effective

  • Electroporation of pre-assembled CRISPR ribonucleoprotein complexes containing crRNA specific for NFIL3 has been successful

• Validation approaches:

  • Confirm knockout efficiency by Western blot analysis (72 hours post-transfection is sufficient to observe protein reduction)

  • Include non-targeting crRNA controls in all experiments

  • Perform functional assays after confirming NFIL3 reduction

This methodology has successfully demonstrated that NFIL3 deletion in differentiated CTLs reduces their killing capacity against target cells .

What controls are essential for NFIL3 overexpression and knockdown studies?

When manipulating NFIL3 expression levels, appropriate controls are critical:

• For overexpression studies:

  • Empty vector controls expressing the same selection marker

  • Overexpression of an unrelated transcription factor of similar size

  • Titration of expression levels to avoid non-physiological artifacts

  • Confirmation of subcellular localization similar to endogenous protein

• For knockdown/knockout studies:

  • Non-targeting guide RNA controls processed through identical protocols

  • Rescue experiments reintroducing NFIL3 expression to confirm specificity

  • Time-course analysis to track protein depletion kinetics

  • Assessment of off-target effects on related family members

In published NFIL3 research, control CRISPR experiments consistently used non-targeting crRNA alongside NFIL3-specific crRNA, allowing for accurate attribution of phenotypic changes to NFIL3 depletion .

How does NFIL3 regulate Regulatory T cell (Treg) function?

NFIL3 plays a critical negative regulatory role in Treg cell function:

• Microarray analysis has shown that Treg cells naturally express lower levels of NFIL3 compared to other CD4+ T cell subsets

• Experimental overexpression of NFIL3 in Treg cells results in:

  • Diminished expression of Foxp3 (the master regulator of Treg cells)

  • Reduced expression of signature Treg genes including Il2ra, Icos, Tnfrsf18, and Ctla4

  • Impaired immunosuppressive activity both in vitro and in vivo

The molecular mechanism involves direct binding of NFIL3 to the Foxp3 gene locus, where it negatively regulates expression. Additionally, NFIL3 induces methylation at regulatory CpG sites in the Foxp3 locus, contributing to the control of Treg cell stability .

This regulatory relationship suggests that maintaining low NFIL3 levels is necessary for proper Treg cell function and immune tolerance.

What role does NFIL3 play in cytotoxic T lymphocyte (CTL) function?

NFIL3 serves as a critical positive regulator of CTL-mediated cytotoxicity:

• Expression dynamics:

  • NFIL3 is upregulated during CTL differentiation

  • Naive CD8+ T cells have undetectable levels of NFIL3

  • Protein levels increase significantly following activation

• Functional impact of NFIL3 deletion:

  • CRISPR-mediated NFIL3 knockout results in approximately 50% reduction in killing capacity against target cells in short-term (3h) assays

  • Reduced killing capacity persists in long-term (8-12h) assays at multiple effector-to-target ratios

• Molecular mechanisms:

  • NFIL3 is not required for immune synapse formation or granule release

  • NFIL3-deficient CTLs show normal conjugate formation with targets and normal centrosome polarization

  • NFIL3 controls the production of cytolytic proteins (perforin and Granzyme B)

  • NFIL3-deficient CTLs show reduced mRNA transcription of both Prf1 and GzmB genes

  • Paradoxically, NFIL3 deletion leads to increased production of TNFα and IFNγ cytokines

These findings demonstrate that NFIL3 plays a cell-intrinsic role in modulating the balance between different cytolytic mechanisms in CTLs.

How can researchers effectively measure changes in NFIL3 expression during T cell activation?

To accurately track NFIL3 expression changes during T cell activation:

• Temporal analysis approach:

  • Isolate naive T cells (CD8+ or CD4+) using magnetic bead selection or flow cytometry sorting

  • Activate cells with appropriate stimuli (anti-CD3/CD28 antibodies or cognate antigen)

  • Collect cells at multiple timepoints (0h, 24h, 48h, 72h, 7 days) post-activation

  • Analyze using Western blot, qPCR, or flow cytometry

• Protein expression analysis:

  • Western blot analysis can detect the gradual increase in NFIL3 protein after activation

  • Flow cytometry with intracellular staining (fixation/permeabilization required) allows for single-cell resolution analysis

  • Subcellular fractionation reveals constitutive nuclear localization pattern

• Transcriptional analysis:

  • qPCR reveals significant upregulation of Nfil3 mRNA between day 0 and day 7 after activation

  • RNA-seq approaches can place NFIL3 expression changes in the context of global transcriptional networks

• Model systems:

  • OT-I TCR transgenic systems (all CD8+ T cells recognize ovalbumin) provide a synchronized activation model for clear temporal analysis

  • In vitro activation with plate-bound CD3/CD28 or antigen-presenting cells both demonstrate NFIL3 upregulation

How should researchers troubleshoot inconsistent NFIL3 antibody staining in flow cytometry?

Inconsistent staining in flow cytometry for NFIL3 can result from several factors:

• Fixation and permeabilization optimization:

  • Test different fixation reagents (formaldehyde, methanol)

  • Compare permeabilization buffers (saponin, Triton X-100)

  • Adjust incubation times and temperatures

  • For nuclear transcription factors like NFIL3, specialized nuclear permeabilization buffers may be required

• Antibody titration:

  • The recommended starting concentration is 0.40 μg per 10^6 cells

  • Perform titration experiments to determine optimal concentration

  • Include positive control cells with known NFIL3 expression (NK92 cells have been validated)

• Signal-to-noise optimization:

  • Include FcR blocking reagents to reduce non-specific binding

  • Extend washing steps to reduce background

  • Use indirect staining with secondary antibodies for signal amplification if needed

• Technical considerations:

  • Ensure consistent handling of all samples

  • Standardize cell numbers across experiments

  • Include isotype controls processed identically to experimental samples

What approaches can resolve contradictory results when studying NFIL3 in different immune cell types?

When faced with contradictory NFIL3 results across different immune cell populations:

• Reconcile expression level discrepancies:

  • NFIL3 expression varies naturally between cell types (lower in Tregs compared to other CD4+ T cells)

  • Consider cell activation status (expression increases after activation in CD8+ T cells)

  • Standardize detection methods across all cell types being compared

• Functional impact analysis:

  • NFIL3 can have opposing roles in different cell types:

    • Negative regulator in Tregs (suppresses Foxp3)

    • Positive regulator in CTLs (promotes cytotoxicity)

  • Perform parallel functional assays appropriate for each cell type

• Context-dependent regulation:

  • Consider the cytokine environment (IL-2, IL-12, TGF-β) which may modify NFIL3 function

  • Examine interaction partners that may differ between cell types

  • Investigate cell-specific epigenetic regulation at the Nfil3 locus

• Technical validation:

  • Use multiple antibody clones targeting different epitopes

  • Employ genetically modified cells (CRISPR knockout) as definitive controls

  • Confirm protein-level observations with transcript analysis

Understanding NFIL3's context-dependent roles requires integrating results from multiple experimental approaches while controlling for technical variables.

What methodologies are recommended for investigating NFIL3 binding to target genes?

To investigate NFIL3's direct binding to target genes:

• Chromatin Immunoprecipitation (ChIP) approach:

  • Use validated ChIP-grade NFIL3 antibodies or epitope-tagged NFIL3 constructs

  • Optimize chromatin fragmentation for transcription factor ChIP (typically 200-500bp fragments)

  • Analyze known target regions like the Foxp3 locus, where NFIL3 has been shown to bind and negatively regulate expression

  • Perform controls with IgG and input chromatin

  • Analyze by quantitative PCR or sequencing (ChIP-seq)

• DNA-binding motif analysis:

  • NFIL3 belongs to the bZIP family of transcription factors

  • Identify putative binding sites using motif analysis software

  • Validate binding using electrophoretic mobility shift assays (EMSA)

  • Perform mutagenesis of predicted binding sites to confirm functionality

• Functional validation:

  • Use reporter assays with wild-type and mutated binding sites

  • Perform genome editing of binding sites to confirm in vivo relevance

  • Correlate binding with transcriptional changes using RNA-seq

NFIL3 has been shown to directly bind and regulate Foxp3 expression, contributing to Treg cell stability control through epigenetic mechanisms . Similar approaches can identify other direct NFIL3 targets in different cell types.

How can researchers investigate the epigenetic mechanisms of NFIL3-mediated gene regulation?

NFIL3 has been shown to influence DNA methylation at target gene loci. To investigate this:

• Bisulfite sequencing approach:

  • Bisulfite sequencing has revealed that NFIL3 induces methylation at regulatory CpG sites in the Foxp3 locus

  • This contributes to control of Treg cell stability and function

  • Researchers should:

    • Compare methylation patterns between wild-type and NFIL3-deficient cells

    • Focus on regulatory regions of genes showing expression changes

    • Use targeted bisulfite sequencing for known NFIL3 targets

• Chromatin accessibility analysis:

  • ATAC-seq or DNase-seq to determine if NFIL3 affects chromatin accessibility

  • Compare accessibility profiles before and after NFIL3 manipulation

  • Correlate with transcriptional changes and methylation status

• Histone modification studies:

  • ChIP-seq for histone marks (H3K4me3, H3K27me3, H3K27ac)

  • Investigate whether NFIL3 binding correlates with specific histone modifications

  • Examine changes in histone modifications after NFIL3 deletion or overexpression

• Integrative analysis:

  • Combine DNA methylation, chromatin accessibility, and histone modification data

  • Correlate with NFIL3 binding sites and gene expression changes

  • Construct models of NFIL3-mediated epigenetic regulation

These approaches can provide comprehensive insights into how NFIL3 influences gene expression through epigenetic mechanisms beyond direct transcriptional regulation.