NINJ2 Antibody

What is NINJ2 and what are its primary biological functions?

NINJ2 (Ninjurin 2) belongs to the ninjurin family of adhesion molecules that mediate cell-to-cell and cell-to-extracellular matrix interactions during development, differentiation, and regeneration of the peripheral nervous system. NINJ2 mRNA is widely expressed in adult human tissues, with highest expression in bone marrow, followed by peripheral leukocytes, lung, and lymph nodes . In the peripheral nervous system, NINJ2 is constitutively expressed in mature sensory and enteric neurons . Recent research has identified NINJ2 as a target of tumor suppressor p53, forming a novel feedback loop where NINJ2 can in turn modulate p53 expression by repressing p53 mRNA translation . Additionally, NINJ2 expression is upregulated after nerve injury in Schwann cells, suggesting a role in nerve regeneration and myelination processes .

What applications are NINJ2 antibodies typically used for?

NINJ2 antibodies are commonly utilized in several experimental applications:

Researchers should note that optimal working dilutions should be determined empirically for each specific experimental setup . For IHC applications, antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 may be used as an alternative .

What tissues or cell types are most suitable for studying NINJ2 expression?

Based on expression patterns, the following tissues and cell types are most suitable for studying NINJ2:

• Peripheral nervous system, particularly sensory and enteric neurons

• Schwann cells, especially after nerve injury

• Bone marrow and peripheral leukocytes (highest expression levels)

• Lung and lymph node tissues

For cell culture studies, researchers have successfully used HEK-293 cells transfected with NINJ2 as a positive control system, as demonstrated in western blot validation studies . Additionally, MCF7 and Molt4 cell lines have been used to study NINJ2 expression in response to DNA damage and p53 activation .

How should I validate the specificity of a NINJ2 antibody in my experimental system?

A comprehensive validation approach for NINJ2 antibodies should include:

• Positive and negative controls: Use NINJ2-transfected cells alongside untransfected cells. Research has validated antibody specificity using "untransfected and NINJ2 transfected HEK-293 cells subjected to SDS-PAGE followed by western blot with mouse anti-NINJ2 monoclonal antibody (1:1000)" .

• Cross-reactivity testing: Since NINJ2 belongs to the ninjurin family, test for cross-reactivity with NINJ1, particularly in experimental systems where both proteins may be expressed.

• Knockout/knockdown validation: Where possible, use NINJ2 knockout or knockdown samples. Published research has utilized SC-specific NINJ2 knockout mice (Dhh cre/+;Ninj2fl/fl) for validation studies .

• Peptide competition: Pre-incubate the antibody with excess NINJ2 immunogen peptide before immunostaining to confirm signal specificity.

• Multiple antibody approach: Use two different antibodies targeting different epitopes of NINJ2 (e.g., one targeting AA 17-81 and another targeting AA 1-142) to confirm consistent detection patterns.

What are the optimal fixation and antigen retrieval methods for NINJ2 immunodetection?

For optimal NINJ2 immunodetection in tissue sections:

Fixation:

• 4% paraformaldehyde is generally suitable for most applications

• For immunofluorescence, avoid prolonged fixation which can mask epitopes

Antigen retrieval:

• Primary recommendation: TE buffer at pH 9.0

• Alternative method: Citrate buffer at pH 6.0

• Heat-induced epitope retrieval (HIER) is typically more effective than enzymatic methods for membrane proteins like NINJ2

Protocol optimization considerations:

• Test multiple antibody dilutions (starting with 1:20-1:200 for IHC and 1:200-1:800 for IF-P)

• Include appropriate blocking steps to reduce background (5% normal serum from the same species as the secondary antibody)

• For dual immunofluorescence studies, carefully select antibody pairs to avoid cross-reactivity

How does NINJ2 interact with the p53 pathway, and what experimental approaches can investigate this relationship?

NINJ2 has been identified as both a target and regulator of p53, forming a novel feedback loop with important implications for cell growth regulation. To investigate this relationship:

• NINJ2 induction by p53: Recent research has demonstrated that NINJ2 can be induced by p53 through a p53-responsive element (p53-RE) in the NINJ2 promoter . This was confirmed through:

  • RT-PCR analysis showing increased NINJ2 expression after doxorubicin treatment in p53-positive cells

  • Western blotting demonstrating elevated NINJ2 protein levels following doxorubicin or camptothecin treatment

  • Chromatin immunoprecipitation (ChIP) assays confirming p53 binding to the NINJ2 promoter, enhanced by doxorubicin treatment

• NINJ2 regulation of p53: Interestingly, NINJ2 can in turn modulate p53 expression by repressing p53 mRNA translation . This bidirectional regulation creates a feedback loop where NINJ2 can influence its own expression through p53.

• Differential effects based on p53 status: The loss of NINJ2 has opposing effects depending on p53 status:

  • In wild-type p53-containing cells: NINJ2 loss inhibits cell growth

  • In mutant p53-containing cells: NINJ2 loss promotes cell growth

To study this relationship experimentally, researchers should consider:

• Using paired cell lines with wild-type versus mutant p53

• Employing NINJ2 knockdown/knockout strategies alongside p53 manipulation

• Conducting polysome profiling to assess NINJ2's effect on p53 mRNA translation

• Utilizing reporter assays with the NINJ2 promoter to quantify p53-dependent transcriptional activation

What experimental approaches can be used to study NINJ2's role in Schwann cell development and myelination?

Based on research indicating NINJ2's upregulation in Schwann cells after nerve injury and its potential role in myelination , these experimental approaches are recommended:

• In vivo models:

  • Utilize SC-specific NINJ2 knockout mice (Dhh cre/+;Ninj2fl/fl) to assess myelination defects

  • Employ nerve injury models to study NINJ2's role in remyelination processes

  • Conduct behavioral tests to evaluate functional outcomes of NINJ2 manipulation

• Imaging techniques:

  • Transmission electron microscopy to visualize myelin ultrastructure

  • Immunofluorescent imaging to assess NINJ2 localization relative to myelin markers

  • Live-cell imaging to monitor dynamic changes during myelination

• Molecular analyses:

  • RNA-Seq to identify NINJ2-dependent pathways in Schwann cells

  • Co-immunoprecipitation to identify NINJ2 binding partners in myelinating Schwann cells

  • Proximity ligation assays to confirm protein-protein interactions in situ

• In vitro models:

  • Establish primary Schwann cell cultures from wild-type and NINJ2-deficient mice

  • Develop co-culture systems with neurons to study myelination processes

  • Utilize advanced 3D culture systems that better recapitulate in vivo nerve architecture

How can I distinguish between NINJ1 and NINJ2 detection in my experimental system?

Distinguishing between these related family members requires careful antibody selection and validation:

• Antibody selection: Choose antibodies raised against regions of minimal homology between NINJ1 and NINJ2. Antibodies targeting amino acids 17-81 of NINJ2 have shown good specificity .

• Western blot discrimination: NINJ1 and NINJ2 have different molecular weights (NINJ2: approximately 16 kDa ), allowing distinction on western blots.

• Control samples:

  • Use tissues with differential expression (NINJ2 is highest in bone marrow, peripheral leukocytes, lung, and lymph nodes )

  • Include samples from NINJ1 or NINJ2 knockout models as controls

  • Test with recombinant NINJ1 and NINJ2 proteins to confirm antibody specificity

• Competitive blocking: Perform parallel staining with pre-incubation using specific blocking peptides for NINJ1 or NINJ2

• Transcript-level analysis: Include RT-PCR or RNA-seq analysis with isoform-specific primers to complement protein-level studies

What are the key considerations for optimizing NINJ2 antibody dilutions in different applications?

Optimization strategies differ by application:

For Western Blotting:

• Start with 1:1000 dilution as demonstrated in published research

• Test a dilution series (e.g., 1:500, 1:1000, 1:2000)

• Include both positive controls (NINJ2-transfected cells) and negative controls

• Optimize blocking conditions to minimize background

For Immunohistochemistry:

• Begin with the manufacturer's recommended range (1:20-1:200)

• Test multiple antigen retrieval methods, comparing TE buffer pH 9.0 with citrate buffer pH 6.0

• Include appropriately fixed positive control tissues (e.g., human tonsillitis tissue)

• Extend incubation times at higher dilutions to maintain signal intensity while reducing background

For Immunofluorescence:

• Start with recommended dilution range (1:200-1:800)

• Optimize fixation conditions and permeabilization steps

• Test signal enhancement systems for detecting low abundance targets

• Use confocal microscopy for better signal-to-noise ratio

Remember that optimal working dilutions should always be determined empirically for each specific experimental system and lot of antibody .

What emerging research areas might benefit from NINJ2 antibody applications?

Several promising research directions could leverage NINJ2 antibodies:

• Cancer biology: The newly discovered feedback loop between NINJ2 and p53 suggests potential roles in tumorigenesis that warrant further investigation . Research could explore NINJ2 as a biomarker in cancers with different p53 status.

• Neuroregeneration: Given NINJ2's upregulation after nerve injury in Schwann cells , antibodies could help map NINJ2 expression patterns during peripheral nerve regeneration and identify potential therapeutic targets.

• Inflammatory processes: With high expression in immune tissues like bone marrow and peripheral leukocytes , NINJ2 may play roles in inflammation or immune regulation that remain to be characterized.

• Developmental neurobiology: NINJ2 antibodies could help trace developmental expression patterns and identify critical periods where NINJ2 functions in nervous system formation.

• Therapeutic targeting: As research progresses, NINJ2 antibodies may help validate this protein as a potential therapeutic target, particularly in conditions involving aberrant myelination or p53 dysfunction.

How might developing technologies enhance NINJ2 antibody applications in research?

Emerging technologies offer new opportunities for NINJ2 research:

• Proximity proteomics (BioID, APEX) could map the NINJ2 interactome in different cellular contexts, revealing new functional associations

• Super-resolution microscopy techniques would allow visualization of NINJ2 distribution at the nanoscale, potentially revealing functional microdomains within the cell membrane

• Single-cell technologies combined with NINJ2 antibodies could identify cell populations with differential NINJ2 expression in heterogeneous tissues

• CRISPR-based screening paired with NINJ2 antibody detection could identify genes that modulate NINJ2 expression or localization

• Tissue clearing and light-sheet microscopy would enable 3D mapping of NINJ2 expression throughout intact tissues, particularly valuable for neural circuits

• Spatially-resolved transcriptomics combined with NINJ2 protein detection could correlate NINJ2 expression with specific transcriptional programs in tissue microenvironments