NFATC3 Antibody

What is NFATC3 and why is it important in research?

NFATC3 (Nuclear Factor of Activated T cells 3) is a transcription factor that plays critical roles in various cellular processes including immune response regulation, cell differentiation, and apoptosis. Also known as NFAT4, NF-AT4c, or NFATX, this protein is approximately 115.6 kilodaltons in mass . NFATC3 is particularly important in research due to its involvement in multiple pathological conditions including cancer progression, inflammatory disorders, and cardiac development. In pancreatic ductal adenocarcinoma (PDAC), for example, hypoxia-induced NFATc3 deSUMOylation enhances tumor progression . In pulmonary research, NFATc3 has been shown to promote inflammation and fibrosis through regulation of chemokine production in macrophages .

How does NFATC3 function at the cellular level?

NFATC3 is primarily located in the cytoplasm in a phosphorylated, inactive state. Upon cellular activation (typically through calcium signaling pathways), calcineurin dephosphorylates NFATC3, exposing its nuclear localization signal . This conformational change enables NFATC3 to translocate to the nucleus where it binds to specific DNA sequences through its highly conserved Rel-like binding domain to regulate gene expression . NFATC3 often forms cooperative complexes with other transcription factors such as AP-1 proteins (Fos and Jun) to modulate target gene expression . This activation pathway is particularly important in T cells, where NFATC3 regulates cytokine production, but also functions in various non-immune cells like smooth muscle and Schwann cells .

What are the known isoforms of NFATC3 and how do they differ?

There are six reported isoforms of NFATC3 with molecular weights ranging from approximately 77.2 to 115.6 kDa . These isoforms are generated through alternative splicing. When working with NFATC3 antibodies, it's important to note that some antibodies, such as clone W15221A, may react with isoforms 1-4 (MW 112.6~115.6 kDa). Additionally, since NFATs are heavily phosphorylated proteins, the observed molecular weights of NFATC3 in experimental settings can be higher than theoretically predicted . This post-translational modification profile adds complexity to the detection and characterization of specific NFATC3 isoforms in research applications.

What criteria should I consider when selecting an NFATC3 antibody for my research?

When selecting an NFATC3 antibody, consider the following criteria:

• Application compatibility: Verify the antibody has been validated for your specific application (WB, IP, IF, IHC, or ELISA) .

• Species reactivity: Ensure the antibody recognizes NFATC3 in your species of interest (human, mouse, rat, etc.) .

• Epitope specificity: Determine which region of NFATC3 the antibody targets and whether it will detect your isoform of interest .

• Validation data: Review published citations, representative images, and validation data provided by the manufacturer .

• Phosphorylation status detection: For functional studies, determine if you need antibodies that differentiate between phosphorylated and non-phosphorylated forms .

• Conjugation needs: Decide if you need unconjugated antibody or one conjugated to fluorophores, enzymes, or other tags based on your experimental design .

• Clone type: Consider whether monoclonal (like F-1 clone) or polyclonal antibodies are more suitable for your application .

How can I validate an NFATC3 antibody for specificity?

To validate NFATC3 antibody specificity:

• Positive and negative controls: Use cell lines or tissues known to express or lack NFATC3 (e.g., Jurkat cells as positive control) .

• Knockdown/knockout verification: Compare antibody signals between wildtype samples and those with NFATC3 knockdown or knockout .

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide to confirm signal specificity.

• Multiple antibody comparison: Use antibodies targeting different epitopes of NFATC3 and compare detection patterns .

• Western blot analysis: Verify that the observed molecular weight matches the expected size of NFATC3 (considering post-translational modifications) .

• Cross-reactivity assessment: Test potential cross-reactivity with other NFAT family members (NFATc1, NFATc2, NFATc4) .

• Phosphorylation-specific validation: For phospho-specific antibodies, treat samples with phosphatases to confirm specificity .

How can I optimize Western blot protocols for NFATC3 detection?

Optimizing Western blot for NFATC3 detection requires attention to several factors:

• Sample preparation:

  • Extract nuclear and cytoplasmic fractions separately to analyze translocation

  • Use phosphatase inhibitors to preserve phosphorylation status

  • Recommended protein amount: 15-20 μg of total cellular lysate

• Gel selection and transfer:

  • Use 7-8% gels for better resolution of high molecular weight NFATC3 (115.6 kDa)

  • Transfer at lower voltage for longer time (e.g., 30V overnight at 4°C) for large proteins

• Antibody concentration:

  • Optimal concentration typically ranges from 0.1-1.0 μg/ml

  • Always titrate to determine the ideal concentration for your specific samples

• Detection considerations:

  • NFATC3 often appears as multiple bands due to phosphorylation states

  • The observed molecular weight may be higher than predicted (112.6-115.6 kDa)

  • For nuclear translocation studies, include both cytoplasmic and nuclear markers as controls

• Signal enhancement:

  • Consider using signal enhancers for low abundance detection

  • For phosphorylated forms, membrane blocking with 5% BSA instead of milk may improve results

What are the best practices for using NFATC3 antibodies in chromatin immunoprecipitation (ChIP) assays?

For optimal ChIP assays with NFATC3 antibodies:

• Crosslinking and chromatin preparation:

  • Use 1% formaldehyde for 10 minutes at room temperature for efficient crosslinking

  • Stop crosslinking with 125 mM glycine for 5 minutes

  • Ensure proper chromatin shearing (200-500 bp fragments) using sonication optimization

• Antibody selection and amount:

  • Consider using a combination of antibodies (such as sc-23814 and sc-1152) to increase efficiency

  • Typically use 2-5 μg of antibody per ChIP reaction

  • Include proper controls: pre-immune serum, IgG, and input controls

• Immunoprecipitation optimization:

  • Pre-clear chromatin with protein A/G beads to reduce background

  • Extend antibody incubation to overnight at 4°C for better binding

  • Perform stringent washing steps to reduce non-specific binding

• Analysis and quantification:

  • Design primers flanking known NFAT binding sites in target genes (e.g., CCR2, TNFα, iNOS promoters)

  • Calculate binding using percent input method or fold enrichment over IgG control

  • Confirm binding site specificity through consensus sequence analysis

• Studying co-binding with other factors:

  • Consider sequential ChIP (re-ChIP) to study co-occupancy with interacting factors like AP-1 or c-Jun

How can I effectively use NFATC3 antibodies for studying nuclear translocation?

To effectively study NFATC3 nuclear translocation:

• Immunofluorescence protocol optimization:

  • Fix cells with 4% paraformaldehyde to preserve subcellular localization

  • Permeabilize with 0.1-0.3% Triton X-100 to allow antibody access

  • Block with 5% normal serum from the species of secondary antibody origin

  • Use anti-NFATC3 antibodies at 1-5 μg/ml concentration

  • Include nuclear counterstain (DAPI or Hoechst)

• Subcellular fractionation approach:

  • Separately extract cytoplasmic and nuclear fractions using appropriate buffers

  • Verify fraction purity using markers (e.g., GAPDH for cytoplasm, Lamin B for nucleus)

  • Analyze NFATC3 distribution by Western blotting of both fractions

• Stimulus conditions for translocation:

  • Induce translocation with calcium mobilizers (ionophores like A23187)

  • PMA/Io (phorbol 12-myristate 13-acetate plus ionophore) treatment is effective for activation

  • Study time-course (15 min to 4 hours) to capture dynamic translocation

• Quantification methods:

  • Use image analysis software to quantify nuclear/cytoplasmic signal ratio

  • Calculate percentage of cells showing nuclear NFATC3 localization

  • Track translocation kinetics through time-course experiments

• Inhibitor studies:

  • Include calcineurin inhibitors (e.g., cyclosporin A) as negative controls

  • Investigate pathway specificity using various inhibitors (calcium chelators, kinase inhibitors)

How do I interpret NFATC3 expression patterns in disease models, especially under hypoxic conditions?

Interpreting NFATC3 expression in disease models requires careful analysis:

• Subcellular localization assessment:

  • Nuclear localization of NFATC3 in tumor specimens correlates with advanced TNM stages in PDAC

  • High nuclear expression often associates with worse patient survival

  • Compare nuclear vs. cytoplasmic staining between normal and pathological tissues

• Hypoxia-specific considerations:

  • Hypoxia induces NFATC3 deSUMOylation via SENP3 upregulation

  • Correlate NFATC3 activation with hypoxia markers like HIF1α expression

  • Consider the relationship between hypoxia duration and NFATC3 activation patterns

• Correlation with disease progression:

  • Analyze NFATC3 nuclear occupancy correlation with clinical features and TNM staging

  • Perform Kaplan-Meier survival analysis based on NFATC3 expression levels

  • Compare expression in primary tumors versus metastatic sites

• Post-translational modification analysis:

  • Assess SUMOylation status of NFATC3 in normal vs. disease states

  • Investigate phosphorylation patterns using phospho-specific antibodies

  • Consider how modifications affect transcriptional activity and target gene expression

What are common problems when using NFATC3 antibodies and how can I solve them?

Common problems and solutions when using NFATC3 antibodies:

• Multiple bands in Western blot:

  • Cause: Post-translational modifications, isoforms, or degradation products

  • Solution: Use phosphatase treatment to confirm phosphorylation states; include protease inhibitors during sample preparation; compare with knockdown controls

• Weak or no signal:

  • Cause: Low NFATC3 expression, inadequate antibody concentration, or inactive antibody

  • Solution: Increase antibody concentration; use signal enhancement; confirm NFATC3 expression in your sample; test positive control samples (Jurkat cells)

• High background in immunostaining:

  • Cause: Non-specific binding, excessive antibody, inadequate blocking

  • Solution: Increase blocking time/concentration; reduce primary antibody concentration; include additional washing steps; try a different blocking agent

• Inconsistent ChIP results:

  • Cause: Inefficient chromatin shearing, antibody binding issues, PCR problems

  • Solution: Optimize sonication conditions; use antibody cocktails ; design multiple primer sets for target regions; include positive control regions

• Difficulty detecting nuclear translocation:

  • Cause: Timing issues, fixation problems, weak activation

  • Solution: Perform time-course experiments; optimize fixation protocol; increase stimulus concentration; use nuclear extraction kits specifically designed for transcription factors

• Inconsistent results between experimental replicates:

  • Cause: Antibody batch variation, sample handling differences, cell state variations

  • Solution: Use the same antibody lot for critical experiments; standardize sample preparation protocols; ensure consistent cell culture conditions

How can I study the role of NFATC3 in the regulation of REDD1 and mTOR signaling?

To investigate NFATC3's role in REDD1-mTOR regulation:

• Genetic manipulation approaches:

  • Use NFATc3 overexpression and knockdown systems to analyze effects on REDD1 expression

  • Employ NFATc3-/- mouse models or CRISPR/Cas9 knockout in cell lines

  • Create phosphorylation or SUMOylation site mutants to study post-translational regulation

• Promoter activity assessment:

  • Clone the REDD1 promoter region (-2931/-97 bp) into a luciferase reporter construct

  • Compare basal and stimulus-induced (PMA/Io) promoter activity with NFATc3 overexpression or knockdown

  • Perform deletion analysis to identify critical NFATc3-responsive elements

• Chromatin binding studies:

  • Conduct ChIP assays to confirm NFATc3 binding to the REDD1 promoter

  • Analyze binding kinetics following various stimuli

  • Investigate co-occupancy with other transcription factors

• mTOR signaling analysis:

  • Monitor mTOR and S6 phosphorylation status following NFATc3 modulation

  • Assess downstream consequences on protein synthesis and autophagy

  • Study the functional connection between NFATc3-REDD1-TSC2 in regulating mTOR activity

• Functional outcome studies:

  • Investigate cellular differentiation markers (e.g., MUC2 for goblet cells)

  • Analyze proliferation, migration, and invasion following NFATc3-REDD1-mTOR manipulation

  • Study the effects of mTOR inhibitors (rapamycin) on NFATc3-mediated functions

How can NFATC3 antibodies be applied to study its role in inflammatory response and acute lung injury?

Advanced applications for studying NFATC3 in inflammatory responses:

• Macrophage activation studies:

  • Compare gene expression profiles (particularly CCR2, TNFα) between wild-type and NFATc3-/- macrophages

  • Use ChIP to analyze NFATc3 binding to inflammatory gene promoters following LPS stimulation

  • Assess NFATc3 nuclear translocation kinetics during inflammatory activation

• In vitro co-culture systems:

  • Establish macrophage-endothelial cell co-cultures to study barrier function

  • Measure endothelial permeability (FITC-dextran flux) when co-cultured with NFATc3-deficient vs. wild-type macrophages

  • Analyze expression of adhesion molecules and tight junction proteins

• In vivo models of acute lung injury:

  • Compare neutrophil infiltration, protein leakage, and arterial oxygenation between NFATc3-/- and wild-type mice in sepsis models

  • Conduct survival studies with and without antibiotic treatment in NFATc3-deficient animals

  • Perform adoptive transfer experiments with NFATc3-deficient macrophages to assess their protective effects

• Multiplex cytokine analysis:

  • Profile the secretome of NFATc3-deficient vs. wild-type cells during inflammatory stimulation

  • Focus on chemokines (CCL2, CXCL2) that regulate inflammatory cell recruitment

  • Correlate cytokine production with nuclear localization of NFATc3

• Therapeutic inhibition studies:

  • Test NFAT inhibitors (beyond cyclosporin A) for effects on inflammatory responses

  • Develop and test cell-specific NFATc3 inhibition strategies

  • Investigate combination therapies targeting both NFATc3 and downstream effectors

What methodologies can I use to investigate the opposing roles of NFATc3 and NFATc4 in cellular apoptosis?

To investigate the opposing roles of NFATc3 and NFATc4 in apoptosis:

• Expression modulation strategies:

  • Compare effects of NFATc3 vs. NFATc4 overexpression on neuronal survival during serum/KCl deprivation

  • Perform knockdown studies to assess differential effects on apoptotic markers

  • Utilize isoform-specific rescue experiments in knockout models

• Target gene analysis:

  • Compare transcriptional profiles regulated by NFATc3 vs. NFATc4

  • Focus on proapoptotic genes (e.g., Trim17) that are differentially regulated

  • Perform ChIP-seq to identify genome-wide binding sites for both factors

• Protein interaction studies:

  • Investigate NFATc3 binding to SUMOylated proteins (e.g., Trim17)

  • Compare protein interactomes of NFATc3 and NFATc4 using mass spectrometry

  • Study competition between NFATc3 and NFATc4 for shared binding partners

• Post-translational modification analysis:

  • Compare SUMOylation patterns between NFATc3 and NFATc4

  • Study how modifications affect their nuclear-cytoplasmic shuttling

  • Investigate modification-dependent protein interactions

• Functional cell death assays:

  • Use live-cell imaging with fluorescent reporters to track apoptosis kinetics

  • Measure caspase activation, mitochondrial membrane potential, and nuclear fragmentation

  • Analyze survival signaling pathways (e.g., BCL-2 family regulation) downstream of each factor

• In vivo models:

  • Compare tissue-specific knockout phenotypes for both factors

  • Analyze development and stress responses in NFATc3-/- vs. NFATc4-/- animals

  • Study double knockout models to assess functional redundancy

How can NFATC3 antibodies be used to study its role in tumor progression and hypoxic adaptation?

Advanced approaches to study NFATc3 in tumor biology:

• Spatial transcriptomics integration:

  • Combine IHC using NFATc3 antibodies with spatial transcriptomics to map NFATc3 activity zones in tumors

  • Correlate NFATc3 nuclear localization with hypoxic regions and expression of hypoxia-response genes

  • Map the distribution of SUMOylated vs. deSUMOylated NFATc3 across tumor microenvironments

• SENP3-NFATc3 axis investigation:

  • Study the kinetics of SENP3-mediated deSUMOylation of NFATc3 under different oxygen tensions

  • Develop antibodies that specifically recognize the SUMOylated K384 site of NFATc3

  • Test SENP3 inhibitors for effects on NFATc3 activity and tumor progression

• Therapeutic targeting strategies:

  • Develop peptide inhibitors that block NFATc3 nuclear translocation

  • Screen for small molecules that modulate NFATc3 SUMOylation

  • Investigate combination approaches targeting both NFATc3 and hypoxia pathways

• Metastasis and invasion models:

  • Analyze NFATc3 target genes involved in cell migration and invasion (e.g., MMP2)

  • Study NFATc3 activity in circulating tumor cells and metastatic deposits

  • Develop 3D organoid systems to assess NFATc3 function in tumor morphogenesis

• Clinical correlation studies:

  • Analyze large patient cohorts for relationships between NFATc3 nuclear localization and response to therapy

  • Develop NFATc3 activity signatures as potential biomarkers for stratifying patients

  • Correlate NFATc3 activity with immune infiltration patterns in the tumor microenvironment

What emerging technologies can enhance NFATC3 antibody applications in single-cell and spatial analysis?

Emerging technologies for advanced NFATc3 research:

• Single-cell protein analysis:

  • Apply mass cytometry (CyTOF) with anti-NFATc3 antibodies to analyze heterogeneity in activation

  • Use single-cell Western blotting to quantify NFATc3 nuclear/cytoplasmic ratios at the individual cell level

  • Implement proximity ligation assays to study NFATc3 interactions in single cells

• Spatial proteomics approaches:

  • Apply multiplexed ion beam imaging (MIBI) or Imaging Mass Cytometry to map NFATc3 localization in tissue context

  • Use Digital Spatial Profiling (DSP) to correlate NFATc3 activity with neighborhood cell types

  • Implement highly multiplexed immunofluorescence to simultaneously visualize NFATc3 with multiple pathway components

• Live-cell imaging innovations:

  • Develop FRET-based sensors to monitor NFATc3 activation in real-time

  • Apply optogenetic tools to precisely control NFATc3 nuclear translocation

  • Use lattice light-sheet microscopy for high-resolution 3D imaging of NFATc3 dynamics

• Genomic integration approaches:

  • Combine CUT&RUN or CUT&Tag with NFATc3 antibodies for high-resolution chromatin binding analysis

  • Apply HiChIP with NFATc3 antibodies to study 3D genome organization at NFATc3 binding sites

  • Implement Calling Cards methods to create a historical record of NFATc3 binding events

• Antibody engineering innovations:

  • Develop nanobodies against NFATc3 for improved penetration in tissue sections

  • Create bifunctional antibodies that can simultaneously detect NFATc3 and its binding partners

  • Engineer antibody fragments for super-resolution microscopy applications