NFATC2 Antibody

What is NFATC2 and what is its biological significance?

NFATC2 (Nuclear Factor of Activated T-cells, Cytoplasmic 2), also known as NFAT1 or NFATp, is a 925 amino acid transcription factor that plays a critical role in regulating immune responses. It exists in a phosphorylated form in the cytoplasm and translocates to the nucleus upon activation via calcineurin-mediated dephosphorylation . This activation-dependent translocation is a key mechanism by which cells distinguish between sustained and transient calcium signals .

NFATC2 is part of a family that includes four NFAT proteins encoded on separate genes: NFAT1 (NFATc2), NFAT2 (NFATc or NFATc1), NFAT3, and NFAT4 (NFATx or NFATc3). These proteins show a low level of sequence similarity with the Dorsal/Rel/NFkB family of transcription factors . NFATC2 is expressed in thymus, spleen, heart, testis, brain, placenta, muscle, and pancreas, indicating its diverse physiological roles beyond immune regulation .

What is the molecular weight of NFATC2 as detected by antibodies in different experimental conditions?

While the calculated molecular weight of NFATC2 is approximately 100 kDa, antibodies typically detect bands at different molecular weights depending on the phosphorylation state:

This difference in molecular weight provides a useful marker for the activation state of NFATC2 in experimental settings . Most commercial antibodies report an observed molecular weight of 135-140 kDa in Western blot applications .

What applications can NFATC2 antibodies be used for in research settings?

NFATC2 antibodies have been validated for multiple experimental applications:

Each application requires specific optimization of antibody dilution and experimental conditions .

How does NFATC2 function in acute myeloid leukemia (AML) pathogenesis?

Recent studies have identified NFATC2 as essential for survival in multiple cytogenetically diverse AML cell lines, revealing a novel mechanism in leukemia pathogenesis . Through RNA-seq and ChIP-seq analyses, NFATC2 has been shown to:

• Maintain cell cycle progression primarily via regulation of CCND1

• Bind to promoter regions of genes involved in oxidative phosphorylation

• Share transcriptional targets with the oncogene c-MYC

• Regulate genes involved in intracellular transport and membrane protein function

The co-ordinated role between NFATC2 and MYC in maintaining AML cell function has been demonstrated through knockdown experiments, where MYC knockdown phenocopied NFATC2 knockdown . ChIP-Seq analysis identified that NFATC2 gene binding targets are enriched with c-Myc DNA consensus binding sequences, and novel NFATC2 DNA binding motifs have also been discovered .

These findings suggest that targeting the NFATC2 pathway could represent a novel therapeutic approach for AML treatment .

What is the relationship between NFATC2 and hypersensitivity reactions in cancer therapy?

NFATC2 plays a critical role in mediating hypersensitivity reactions to chemotherapeutic agents such as L-asparaginase (ASNase) used in acute lymphoblastic leukemia treatment . Genetic or pharmacological inhibition of NFATC2 provides protection from these hypersensitivity reactions through multiple mechanisms:

• Reduction in antigen-specific and total IgE levels

• Increase in CD4+ regulatory T cells

• Decrease in CD4+ IL-4+ cells

• Elevation of IL-10/TGF-β1 levels

• Reduction in IL-4/IL-13 levels

• Decreased FcεRI expression on basophils and mast cells

• Reduced IgE-mediated mast cell activation

The protective effect operates by attenuating Th2 immune responses after antigen exposure, which limits the anaphylaxis-promoting responses of B cells, basophils, mast cells, and vascular endothelial cells . These findings suggest NFATC2 inhibition as a potential therapeutic strategy to mitigate unwanted immune/hypersensitivity responses against ASNase and possibly other therapeutic agents .

How does NFATC2 contribute to inflammatory bowel disease (IBD) pathogenesis?

Significantly higher expression of NFATC2 has been found in ulcerative colitis tissues compared to control samples, suggesting a role in IBD pathogenesis . Mechanistically, NFATC2 mediates its pathogenic effects through:

• Regulation of T cell-dependent experimental colitis

• Control of IL-6-dependent T cell activation

• Influence on T cell apoptosis in the lamina propria

• Modulation of cytokine production by mucosal T cells

Interestingly, NFATC2-deficient T cells produced significantly lower amounts of pro-inflammatory cytokines (IFN-γ, IL-6, IL-17) but higher amounts of IL-4 than wild-type T cells . Additionally, colonic lamina propria mononuclear cells from NFATC2-deficient mice produced significantly lower amounts of IL-6, IL-13, and IL-17 in experimental colitis .

These findings establish NFATC2 as a potential therapeutic target for inflammatory bowel diseases by modulating T cell responses and cytokine production in intestinal inflammation .

What are the optimal conditions for using NFATC2 antibodies in Western blot applications?

For optimal Western blot results with NFATC2 antibodies, consider the following protocol parameters:

By Western blot, antibodies like MA1-025 detect distinct bands representing phosphorylated NFATC2 (~140 kDa) in resting immune cells and dephosphorylated NFATC2 (~120 kDa) in stimulated cells .

For immunofluorescence (IF) applications using NFATC2 antibodies, researchers should consider:

For studying NFATC2 translocation between cytoplasm and nucleus, stimulation conditions (e.g., calcium ionophores, PMA/ionomycin) should be carefully optimized to capture the dynamic shuttling process .

How can researchers distinguish between phosphorylated and dephosphorylated forms of NFATC2?

The phosphorylation state of NFATC2 is critical for its function and cellular localization. Researchers can distinguish between these forms through several approaches:

• Western Blot Analysis: Phosphorylated NFATC2 migrates at ~140 kDa while dephosphorylated NFATC2 appears at ~120 kDa .

• Subcellular Localization:

  • Phosphorylated form: predominantly cytoplasmic

  • Dephosphorylated form: nuclear localization

• Experimental Manipulation:

  • To observe predominantly phosphorylated form: use resting immune cells or serum-starved conditions

  • To observe dephosphorylated form: stimulate cells with calcium ionophores, PMA/ionomycin, or anti-CD3/CD28 antibodies

• Phosphatase Treatment Control: Treat lysates with lambda phosphatase to confirm band shifts are due to phosphorylation

Phosphorylation status serves as both a functional readout and an experimental tool for studying NFATC2 activation in different cellular contexts .

What controls should be included when validating NFATC2 antibody specificity?

To ensure the specificity and reliability of NFATC2 antibody results, researchers should include the following controls:

Additionally, the MA1-025 antibody does not cross-react with NFAT2 (NFATc, NFATc1), which is important for distinguishing between NFAT family members in experimental settings .

How can researchers troubleshoot unexpected results in NFATC2 protein detection?

When encountering unexpected results with NFATC2 antibodies, consider these troubleshooting approaches:

For particularly challenging samples, consider using phosphorylation-specific antibodies if available, or combining multiple detection methods to confirm results .

What new findings have emerged regarding NFATC2's role in cell cycle regulation?

Recent studies have revealed that NFATC2 plays a critical role in maintaining cell cycle progression in human AML cells, primarily through regulation of CCND1 . This finding expands our understanding of NFATC2 beyond its traditional role in immune cell activation.

Key research developments include:

• NFATC2 has been identified as a novel binding and transcriptional target of histone lysine demethylase 4A (KDM4A) in AML cells .

• Cytogenetically diverse AML cell lines show dependency on NFATC2 for colony formation in vitro, highlighting its role in leukemia cell survival .

• Global transcriptome profiling has identified genes regulated by NFATC2 that are involved in:

  • Cell cycle control

  • Oxidative phosphorylation

  • Intracellular transport

  • Membrane protein function

• NFATC2 and c-MYC share transcriptional targets, with MYC knockdown phenocopying NFATC2 knockdown, suggesting coordinated roles in maintaining leukemia cell function .

These findings indicate that NFATC2 regulation extends beyond immune responses to fundamental cellular processes like proliferation and metabolism .

How does NFATC2 interact with mitochondrial function in cancer cells?

An emerging area of NFATC2 research concerns its role in regulating mitochondrial function, particularly in cancer cells:

Through RNA-seq and ChIP-seq analyses, NFATC2 has been shown to bind to the promoter regions of genes involved in oxidative phosphorylation and subsequently regulate their expression in AML cells . This finding establishes a previously unrecognized connection between NFATC2 and cellular energy metabolism.

The MYC-NFATC2 axis appears to maintain both cell cycle progression and mitochondrial function in AML cells, suggesting a coordinated role in cancer cell bioenergetics . This relationship provides new insights into how transcription factors traditionally associated with immune function may contribute to metabolic reprogramming in cancer.

These discoveries open new avenues for therapeutic intervention by potentially targeting both proliferative and metabolic vulnerabilities in cancer cells dependent on NFATC2 signaling .

What are the emerging therapeutic applications targeting NFATC2 in immune-mediated diseases?

Recent research has identified NFATC2 as a promising therapeutic target for various immune-mediated diseases:

• Hypersensitivity Reactions: Genetic or pharmacological inhibition of NFATC2 provides protection from hypersensitivity reactions to chemotherapeutic agents like L-asparaginase through attenuation of Th2 immune responses .

• Inflammatory Bowel Disease: NFATC2 mediates T cell-dependent experimental colitis, with NFATC2-deficient mice showing protection from disease development. Targeting NFATC2 could potentially modulate intestinal inflammation .

• Acute Myeloid Leukemia: Multiple AML cell lines show dependency on NFATC2 for survival, suggesting it as a potential therapeutic target in leukemia treatment .

Experimental approaches using the NFAT inhibitor 11R-VIVIT have shown protection from anaphylaxis and attenuation of Th2 responses in mouse models . This pharmacological validation provides proof-of-concept for NFATC2 inhibition as a therapeutic strategy.

These findings collectively suggest that NFATC2 inhibition could represent a novel approach for treating conditions ranging from allergic reactions to inflammatory disorders and certain cancers .