NFATC1 Antibody

What is NFATC1 and what are its key biological functions?

NFATC1 (Nuclear Factor of Activated T cells c1, also known as NFAT2) is a transcription factor primarily expressed in T cells and mast cells. It serves as a critical regulator of cytokine transcription and immune responses . In its inactive state, NFATC1 is phosphorylated and localized in the cytoplasm, particularly at Ser172 . Following cellular activation and calcium influx, NFATC1 undergoes dephosphorylation by calcineurin, which facilitates its translocation to the nucleus . Within the nucleus, NFATC1 forms complexes with AP-1 proteins like Fos and Jun, binding to specific DNA sequences to promote cytokine gene transcription in T cells . Beyond immune regulation, NFATC1 plays essential roles in cardiovascular development, osteoclast differentiation affecting bone resorption, and dysregulation of NFATC1 activity has been implicated in autoimmune disorders and cardiac hypertrophy .

How is NFATC1 activated and regulated in cellular systems?

NFATC1 activation follows a calcium-dependent signaling pathway. Increased intracellular calcium concentrations trigger calcineurin, a phosphatase that dephosphorylates NFATC1, enabling its nuclear translocation . This process is essential for T cell activation and effector functions . The pathway involves multiple regulatory mechanisms: Akt signaling inhibits GSK3, which in turn promotes NFATC1 activation . In VEGF-stimulated endothelial cells, NFATC1 activation leads to genome-wide binding to target genes with preferential binding to H3K4me3-positive active promoter regions, as revealed by ChIP-seq analysis . This dynamic binding pattern is critical for endothelial cell activation in response to VEGF . Additionally, NFATC1 activation is targeted by immunosuppressive drugs in transplantation medicine, highlighting its clinical relevance in immune regulation .

What evidence supports NFATC1's role in anti-tumor immunity?

Research using targeted deletion of NFATC1 in T cells (NFATc1ΔCD4 model) has demonstrated its crucial function in anti-tumor immune responses, particularly in non-small cell lung cancer (NSCLC) . Mice lacking NFATC1 in T cells exhibited increased lung tumor growth, associated with impaired T cell activation and function . The absence of NFATC1 led to reduced IL-2 production, which negatively influenced the development of memory CD8+ T cells . Furthermore, researchers observed a reduction in effector memory T cells (TEM) and CD103+ tissue-resident memory T cells (TRM) in the lungs of tumor-bearing NFATc1ΔCD4 mice, indicating compromised cytotoxic T cell responses and reduced tissue-homing capacity . The study also found that PD-1 blockade therapy significantly induced NFATC1 in T cells, accompanied by increased anti-tumor cytotoxic functions, suggesting NFATC1 as a key mediator in checkpoint inhibitor immunotherapy responses .

What criteria should researchers consider when selecting an NFATC1 antibody?

When selecting an NFATC1 antibody, researchers should evaluate several critical parameters for experimental success. First, consider the species reactivity needed for your model system—available antibodies demonstrate varying reactivity profiles with mouse, rat, and human NFATC1 . Application compatibility is equally important, as different research questions require specific detection methods. For instance, the NFATc1 monoclonal antibody (7A6) is validated for Western blotting, immunoprecipitation, immunofluorescence, immunohistochemistry on paraffin sections, and flow cytometry . For human NFATC1 detection, antibodies like the goat anti-human NFATC1 have been validated specifically for Western blot applications in cell lines such as Jurkat, Ramos, and Raji . The antibody's target epitope location can influence detection of specific NFATC1 isoforms or phosphorylation states—for example, some antibodies target amino acids 197-304 of human NFATC1 . Finally, consider the antibody format (unconjugated or directly conjugated with fluorophores or enzymes) based on your detection system requirements.

How can researchers validate NFATC1 antibody specificity and performance?

Proper validation of NFATC1 antibodies is essential for generating reliable research data. A comprehensive validation approach includes multiple methods. Western blotting serves as a primary validation tool, where antibodies should detect NFATC1 at the expected molecular weight of approximately 110-120 kDa . Researchers should test the antibody in relevant positive control lysates such as Jurkat (human acute T cell leukemia), Ramos or Raji (human Burkitt's lymphoma) cell lines . For advanced validation, Simple Western™ technology can provide automated, size-based detection with higher sensitivity . Immunoprecipitation followed by mass spectrometry can confirm the identity of the precipitated protein. For functional validation, genetic approaches using NFATC1 knockout or knockdown models provide the most stringent control. The antibody should show significantly reduced or absent signal in these models. Cross-reactivity testing with related NFAT family members (NFATC2, NFATC3, NFATC4) is also important to ensure specificity within this conserved protein family.

What are the optimal storage and handling conditions for NFATC1 antibodies?

Proper storage and handling of NFATC1 antibodies are crucial for maintaining their functionality and experimental reproducibility. NFATC1 antibodies should be stored at -20°C to -70°C for long-term preservation (up to 12 months from the date of receipt) in a manual defrost freezer . After reconstitution, the antibody can be stored at 2-8°C under sterile conditions for approximately one month . For extended storage post-reconstitution, aliquot the antibody and store at -20°C to -70°C for up to six months under sterile conditions . It is essential to avoid repeated freeze-thaw cycles, which can lead to protein denaturation and reduced antibody efficacy . When working with conjugated antibodies (fluorophore-labeled or enzyme-linked), protect them from prolonged exposure to light to prevent photobleaching. Before each use, centrifuge the antibody solution briefly to collect the liquid at the bottom of the vial and ensure accurate pipetting. For reconstitution, follow manufacturer-specific guidelines for the appropriate buffer composition to maintain antibody stability and function.

What are the optimal conditions for using NFATC1 antibodies in Western blotting?

For optimal Western blot detection of NFATC1, researchers should consider several critical parameters. Sample preparation should include appropriate lysis buffers containing phosphatase inhibitors to preserve the native phosphorylation state of NFATC1, which affects its molecular weight and detection. When loading samples, 20-50 μg of total protein per lane typically yields good results. NFATC1 appears as bands between 110-120 kDa under reducing conditions . For membrane transfer, PVDF membranes are recommended over nitrocellulose for better protein retention . Blocking should be performed with 5% non-fat dry milk or BSA in TBST. For primary antibody incubation, a concentration of 1 μg/mL has been validated for the goat anti-human NFATC1 antibody , though optimal concentrations may vary by manufacturer and should be determined empirically. Incubation overnight at 4°C typically yields the best signal-to-noise ratio. For detection, HRP-conjugated secondary antibodies specific to the host species of the primary antibody (e.g., HRP-conjugated anti-goat IgG for goat primary antibodies) provide reliable results . Enhanced chemiluminescence (ECL) detection systems offer appropriate sensitivity for NFATC1 visualization.

How can NFATC1 antibodies be effectively used in immunohistochemistry and immunofluorescence?

For successful immunohistochemical detection of NFATC1, tissue fixation and antigen retrieval are critical first steps. Paraffin-embedded sections require appropriate antigen retrieval methods, typically using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) with heat treatment. For immunofluorescence applications, cells should be fixed with 4% paraformaldehyde followed by permeabilization with 0.1-0.5% Triton X-100. Blocking with 5-10% normal serum from the same species as the secondary antibody helps reduce background. Primary antibody dilutions typically range from 1:50 to 1:200 for immunohistochemistry and immunofluorescence, though optimal concentrations should be determined empirically for each antibody and tissue type. For NFATC1, nuclear localization indicates the activated form, while cytoplasmic staining represents inactive NFATC1 . This subcellular localization pattern provides valuable information about NFATC1 activation status and can be used as a readout in functional studies. For immunofluorescence, direct conjugates of NFATC1 antibodies with fluorophores (FITC, PE, or various Alexa Fluor dyes) are commercially available , enabling multiplexed staining with other markers.

What are the key considerations for NFATC1 detection in flow cytometry?

Flow cytometric analysis of NFATC1 requires specific considerations due to its role as a transcription factor with different localization patterns depending on activation status. For successful detection, cells must be fixed and permeabilized using commercially available kits specifically designed for nuclear factors. The BD Cytofix/Cytoperm™ kit or eBioscience Foxp3 staining buffer set are suitable options as they enable access to nuclear epitopes. For optimal staining, use fluorochrome-conjugated NFATC1 antibodies such as PE, FITC, or Alexa Fluor conjugates . The antibody concentration for flow cytometry typically ranges from 2-5 μg/mL or follows manufacturer recommendations for pre-conjugated antibodies. Since NFATC1 shows dynamic translocation between cytoplasm and nucleus upon activation, researchers should consider the activation state of cells and the timing of analysis after stimulation. For quantitative assessment, it's essential to include appropriate isotype controls matched to the primary antibody's host species and isotype (e.g., mouse IgG1κ for the 7A6 clone) . When studying NFATC1 in T cells, consider co-staining with surface markers like CD3, CD4, and CD8 before fixation and permeabilization to identify specific T cell subsets.

What are common problems encountered when using NFATC1 antibodies and how can they be resolved?

Researchers working with NFATC1 antibodies may encounter several common issues. Weak or absent signals in Western blots often result from insufficient antibody concentration or inadequate sample preparation. If this occurs, increase protein loading to 50-100 μg per lane, optimize antibody concentration, extend incubation time, or enhance detection sensitivity with more sensitive chemiluminescent substrates. Multiple bands on Western blots may represent different NFATC1 isoforms, post-translational modifications, or non-specific binding. To address this, use more stringent washing conditions, optimize blocking procedures, or validate with alternative antibodies targeting different epitopes of NFATC1. High background in immunohistochemistry or immunofluorescence often stems from insufficient blocking or excessive antibody concentration. Improve results by extending blocking time with 5-10% serum, titrating antibody concentration, and including additional washing steps. For flow cytometry, poor discrimination between positive and negative populations may occur due to inadequate permeabilization. Optimize by testing different permeabilization reagents and ensure samples are properly fixed. If NFATC1 signal is inconsistent between experiments, standardize cell activation conditions, as NFATC1 localization and expression are highly dependent on activation status .

How should researchers interpret variations in NFATC1 detection across different experimental systems?

Interpretation of NFATC1 detection requires careful consideration of biological and technical variables. The molecular weight of NFATC1 may vary from 110-120 kDa depending on the cell type and activation state . This variation reflects post-translational modifications, particularly phosphorylation status, which changes upon cellular activation . Subcellular localization provides critical functional information: cytoplasmic NFATC1 generally indicates an inactive, phosphorylated state, while nuclear localization suggests activation following dephosphorylation by calcineurin . When comparing NFATC1 expression across different experimental models, consider species-specific differences in NFATC1 sequence and regulation. For instance, while the NFATc1 monoclonal antibody (7A6) recognizes mouse, rat, and human NFATC1 , subtle differences in epitope affinity may affect signal intensity. Expression levels naturally vary between cell types, with highest expression typically observed in activated T cells, mast cells, and certain cancer cell lines like Jurkat . During data analysis, normalize NFATC1 signal to appropriate loading controls for protein quantity comparisons, and include positive control samples (e.g., stimulated Jurkat cells) to validate detection systems across experiments.

How can researchers differentiate between NFATC1 isoforms and phosphorylation states?

Distinguishing between NFATC1 isoforms and phosphorylation states requires specialized approaches. NFATC1 exists in multiple splice variants, and its activity is regulated by extensive phosphorylation. To differentiate isoforms, researchers should select antibodies targeting isoform-specific regions or employ RT-PCR with isoform-specific primers. For identifying phosphorylation states, phospho-specific antibodies that recognize NFATC1 when phosphorylated at specific residues (particularly Ser172, a key regulatory site ) are valuable tools. Alternatively, researchers can use lambda phosphatase treatment of cell lysates before Western blotting—a mobility shift to lower molecular weight after treatment indicates the presence of phosphorylated NFATC1. Two-dimensional gel electrophoresis can separate NFATC1 based on both molecular weight and isoelectric point, helping distinguish phosphorylated forms. For functional studies, researchers can use calcineurin inhibitors (cyclosporine A or FK506) to maintain NFATC1 in its phosphorylated state, or calcium ionophores (ionomycin) combined with PKC activators (PMA) to induce dephosphorylation. The subcellular localization observed in immunofluorescence or cell fractionation studies provides additional information—cytoplasmic localization suggests phosphorylated (inactive) NFATC1, while nuclear localization indicates dephosphorylated (active) forms .

How can NFATC1 antibodies be utilized in ChIP-seq experiments to identify genome-wide binding patterns?

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) with NFATC1 antibodies provides valuable insights into genome-wide NFATC1 binding patterns and gene regulation mechanisms. For successful ChIP-seq experiments, researchers should select ChIP-grade NFATC1 antibodies with validated specificity and low background. Crosslinking conditions must be optimized to capture transient NFATC1-DNA interactions, typically using 1% formaldehyde for 10-15 minutes at room temperature. Sonication parameters should be carefully adjusted to generate DNA fragments of 200-500 bp for optimal sequencing resolution. Research has shown that in VEGF-stimulated endothelial cells, predominant NFATc1-occupied genomic regions overlap with promoter-associated histone marks, particularly H3K4me3-positive sites . This finding suggests that NFATC1 preferentially binds to active promoter regions . When analyzing ChIP-seq data, comparison with epigenetic histone marks helps contextualize NFATC1 binding within the chromatin landscape . For validation of novel NFATC1 target genes identified through ChIP-seq, researchers should perform targeted ChIP-PCR followed by functional studies using gene knockdown or overexpression approaches. Notable NFATC1 target genes discovered through ChIP-seq include CXCR7 and RND1, which regulate endothelial cell migration, tube formation, and barrier function .

What insights do genetic studies of NFATC1 polymorphisms provide for translational research?

Genetic studies of NFATC1 polymorphisms have revealed important associations with clinical outcomes, particularly in transplantation medicine. A comprehensive analysis using target sequencing identified multiple single nucleotide polymorphisms (SNPs) in the NFATC1 gene that are significantly associated with biopsy-proven acute rejection (BPAR) in renal transplant recipients . After adjusting for clinical confounding factors, four NFATC1 SNPs—rs2290154, rs2304738, rs754093, and rs754096—showed statistically significant distribution differences between stable allograft function and BPAR groups . Among these, rs2290154 demonstrated a particularly strong association with rejection severity, correlating significantly with Banff scores and renal tubulitis . Functional studies of rs2290154 revealed its mechanistic impact: the mutant variant remarkably promoted T cell proliferation, increased NFATC1 mRNA transcription and protein expression, and enhanced interleukin-2 (IL-2) secretion . This indicates that genetic variations in NFATC1 can directly influence T cell activation and proliferation, affecting immune responses in transplantation. The association between NFATC1 polymorphisms and transplant rejection provides potential biomarkers for personalized risk assessment and could inform individualized immunosuppressive regimens to prevent rejection episodes based on patient genotypes.

How does NFATC1 contribute to the anti-tumor immune response, and how can this be studied?

NFATC1 plays a multifaceted role in anti-tumor immunity that can be investigated through various experimental approaches. Studies using conditional knockout mice with T cell-specific deletion of NFATC1 (NFATc1ΔCD4) have demonstrated that NFATC1 is indispensable for effective anti-tumor immune responses, particularly in non-small cell lung cancer (NSCLC) . In these models, the absence of NFATC1 led to increased tumor growth associated with impaired T cell activation and function . To study NFATC1's role in tumor-infiltrating lymphocytes, researchers can employ flow cytometry with NFATC1 antibodies to assess activation status, combined with T cell functional markers. Immunohistochemistry of tumor sections allows visualization of NFATC1 nuclear translocation in various immune cell subsets within the tumor microenvironment. NFATC1 regulates memory CD8+ T cell development, particularly effector memory T cells (TEM) and CD103+ tissue-resident memory T cells (TRM), which are critical for sustained anti-tumor responses . These populations can be characterized using flow cytometry with appropriate surface markers combined with NFATC1 staining. Importantly, NFATC1 may serve as a key mediator in checkpoint inhibitor therapy responses, as PD-1 blockade significantly induces NFATC1 in T cells, accompanied by enhanced anti-tumor cytotoxic functions . This relationship can be studied by treating tumor-bearing mice or human samples with anti-PD-1 antibodies and measuring changes in NFATC1 expression and localization.

What are the latest discoveries about NFATC1's role in endothelial cell regulation and angiogenesis?

Recent research has uncovered NFATC1's significant involvement in endothelial cell regulation and angiogenesis through genome-wide binding studies. ChIP-seq analysis of VEGF-stimulated primary endothelial cells revealed that NFATC1 preferentially binds to H3K4me3-positive active promoter regions . This strategic binding pattern suggests that NFATC1 targets genes already in a transcriptionally permissive chromatin state. Through these comprehensive genomic analyses, researchers identified novel NFATC1-regulated genes, including CXCR7 and RND1, which have multiple functions in endothelial cell biology . Functional studies demonstrated that CXCR7 knockdown significantly impaired SDF-1- and VEGF-mediated cell migration and tube formation, indicating its crucial role in angiogenic responses . Similarly, RND1 silencing disrupted vascular barrier function, caused RhoA hyperactivation, and further stimulated VEGF-mediated vascular outgrowth from aortic rings . These findings establish that dynamic NFATC1 binding to target genes is critical for VEGF-mediated endothelial cell activation and subsequent angiogenic processes . The VEGF-calcineurin-NFAT signaling axis has also been implicated in tumor angiogenesis and metastasis, as demonstrated in Down syndrome model mice and clinical patient samples, highlighting potential therapeutic targets in cancer treatment .

How does NFATC1 function in immune cell memory formation, and what are the implications for immunotherapy?

NFATC1 plays a critical role in the formation and maintenance of memory T cells, with significant implications for cancer immunotherapy. Studies using mice with T cell-specific NFATC1 deletion (NFATc1ΔCD4) revealed that NFATC1 is essential for the development of memory CD8+ T cell subsets . In the absence of NFATC1, reduced IL-2 production negatively influenced memory CD8+ T cell development . Specifically, researchers observed significant reductions in effector memory T cells (TEM) and CD103+ tissue-resident memory T cells (TRM) in the lungs of tumor-bearing NFATc1ΔCD4 mice . This reduction was associated with impaired cytotoxic T cell responses and diminished tissue-homing capacity . The connection between NFATC1 and T cell exhaustion represents another critical aspect of its function in anti-tumor immunity. In NFATc1ΔCD4 mice, PD-1 was found co-expressed with CD4+ICOS+ T cells in tumor-draining lymph nodes, correlating with enhanced tumor growth . Importantly, PD-1 blockade therapy significantly induced NFATC1 expression in T cells, accompanied by increased anti-tumor cytotoxic functions . These findings suggest that NFATC1 may serve as a key mechanistic link in checkpoint inhibitor therapy responses, potentially explaining variability in clinical outcomes. Understanding these pathways could inform strategies to enhance immunotherapy efficacy through NFATC1 modulation.

What emerging technologies and approaches are advancing our understanding of NFATC1 biology?

The field of NFATC1 research is benefiting from several cutting-edge technologies that provide unprecedented insights into its function. Single-cell technologies, including single-cell RNA-seq combined with protein detection (CITE-seq), allow researchers to correlate NFATC1 expression and activation with comprehensive cellular phenotypes at individual cell resolution. This approach is particularly valuable for understanding NFATC1's role in heterogeneous immune cell populations within complex microenvironments like tumors or inflamed tissues. CRISPR-Cas9 gene editing enables precise modification of NFATC1 or its target genes to investigate functional consequences in various cellular contexts. CRISPR screens can identify novel regulators of the NFATC1 pathway or downstream effectors. Advanced imaging techniques such as lattice light-sheet microscopy with NFATC1-fluorescent protein fusions permit real-time visualization of NFATC1 nuclear translocation dynamics in living cells following activation. Proximity labeling methods like BioID or APEX2 fused to NFATC1 can identify novel interaction partners in specific subcellular compartments or activation states. Target sequencing approaches have already proven valuable in identifying NFATC1 SNPs associated with clinical outcomes in transplantation, as demonstrated in studies examining biopsy-proven acute rejection in renal transplant recipients . Integration of genomic, transcriptomic, and proteomic data through systems biology approaches will continue to advance our understanding of NFATC1's complex role in diverse physiological and pathological processes.

NFATC1 Antibody Applications Table