Phospho-NFATC3 (Ser165) Antibody

What is Phospho-NFATC3 (Ser165) Antibody and what specifically does it detect?

Phospho-NFATC3 (Ser165) Antibody is a highly specific antibody designed to detect Nuclear Factor of Activated T-cells, cytoplasmic 3 (NFATc3) protein only when phosphorylated at serine residue 165. The antibody recognizes a specific amino acid sequence (typically 131-180 aa region) surrounding the phosphorylated Ser165 site . This antibody serves as a valuable research tool for studying the phosphorylation-dependent regulation of NFATc3, which is critical for understanding its role in various cellular processes including T-cell activation, calcium signaling responses, and pathological conditions such as cancer progression .

The specificity is achieved through the immunogen design - the antibody is generated against a synthesized phosphopeptide derived from human NFAT4 (another name for NFATc3) around the Ser165 phosphorylation site . This ensures that the antibody only binds to NFATc3 when this specific residue is phosphorylated, making it an excellent tool for monitoring the phosphorylation status of NFATc3 at Ser165 in experimental settings.

What are the recommended applications and dilutions for Phospho-NFATC3 (Ser165) Antibody?

Phospho-NFATC3 (Ser165) Antibody has been validated for multiple applications with specific dilution recommendations for optimal results:

For reproducible results, researchers should optimize these dilutions for their specific experimental conditions and sample types. Validation images from multiple sources demonstrate successful application in HeLa cells for Western blot and immunofluorescence, as well as in human liver cancer tissue sections for immunohistochemistry .

What is the biological significance of NFATc3 Ser165 phosphorylation?

The phosphorylation status of NFATc3 at Ser165 plays a crucial role in regulating its subcellular localization and transcriptional activity. Key findings include:

• Regulation of subcellular localization: Heavily phosphorylated NFATc3 (including at Ser165) resides in the cytoplasm of resting cells. Upon dephosphorylation, NFATc3 translocates to the nucleus to activate target gene expression .

• Kinetics of activation: NFATc3 undergoes rapid dephosphorylation and nuclear translocation, typically complete within 20 minutes of stimulation, which is significantly faster than other NFAT family members like NFATc4 .

• Response to calcium signaling: Phosphorylation at Ser165 is responsive to calcium-dependent signaling cascades. Treatment of cells with calcium (40nM for 30 minutes) has been shown to alter the phosphorylation state of NFATc3 at Ser165 .

• Disease relevance: Abnormal phosphorylation of NFATc3 has been implicated in several pathological conditions, particularly in pancreatic ductal adenocarcinoma (PDAC) where excessive activation correlates with advanced stages and shorter patient survival time .

Understanding the dynamics of Ser165 phosphorylation provides insights into the molecular mechanisms controlling NFATc3-mediated transcriptional regulation in both normal physiology and disease states.

How can I validate the specificity of Phospho-NFATC3 (Ser165) Antibody?

Validating antibody specificity is crucial for ensuring reliable experimental results. For Phospho-NFATC3 (Ser165) Antibody, consider these methodological approaches:

• Phospho-peptide blocking: Perform Western blot or immunostaining with and without pre-incubation of the antibody with the phosphorylated peptide used as the immunogen. The specific signal should be blocked by the phospho-peptide but not by the non-phosphorylated version .

• Phosphatase treatment: Treat half of your samples with lambda phosphatase before immunoblotting. The signal should disappear in phosphatase-treated samples if the antibody is truly phospho-specific .

• Stimulation/inhibition experiments: Treat cells with stimuli known to induce NFATc3 phosphorylation (e.g., calcium) or inhibitors of phosphorylation (e.g., calcineurin inhibitors like Cyclosporin A) . The phospho-signal should change accordingly.

• Knockdown/knockout controls: Use siRNA or CRISPR/Cas9 to reduce or eliminate NFATc3 expression. The phospho-specific signal should be reduced or eliminated in these samples .

Published validation data shows that in Western blot analysis of HeLa cell lysates treated with calcium (40nM for 30 minutes), the Phospho-NFATC3 (Ser165) Antibody detects a specific band at approximately 115kDa, which can be blocked by the phospho-peptide, confirming its phospho-specificity .

What controls should I include when using Phospho-NFATC3 (Ser165) Antibody in my experiments?

Proper controls are essential for interpreting results with phospho-specific antibodies. For experiments using Phospho-NFATC3 (Ser165) Antibody, include:

• Positive controls:

  • Cell lines known to express phosphorylated NFATc3 (e.g., HeLa cells treated with calcium)

  • Tissues known to express phosphorylated NFATc3 (e.g., thymus, skeletal muscle)

  • Internal loading control (e.g., GAPDH) for normalization in Western blots or cell-based assays

• Negative controls:

  • Samples treated with lambda phosphatase to remove phosphorylation

  • Secondary antibody alone (no primary antibody)

  • Non-specific IgG of the same species as the primary antibody

  • NFATc3 knockout or knockdown samples

• Comparative controls:

  • Parallel detection with antibody against total NFATc3 (non-phospho-specific) to normalize phospho-signal to total protein levels

  • Treatment with phosphatase inhibitors to increase phosphorylation

  • Treatment with kinase inhibitors to decrease phosphorylation

For cell-based phosphorylation ELISAs, the manufacturer recommends both positive controls (anti-GAPDH antibody) and negative controls (HRP-conjugated secondary antibodies alone) performed in the same plate as the phospho-NFATc3 target experiments, with all conditions performed in duplicate or triplicate for accuracy .

How can I optimize sample preparation for detecting phosphorylated NFATc3?

Detecting phosphorylated proteins requires careful sample preparation to preserve phosphorylation status. Follow these methodological steps for optimal results with Phospho-NFATC3 (Ser165) Antibody:

• Cell/tissue lysis:

  • Use ice-cold lysis buffers containing phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate)

  • Include protease inhibitors to prevent protein degradation

  • Perform lysis quickly and keep samples cold throughout processing

• For Western blotting:

  • Use freshly prepared samples when possible

  • Heat samples at 95-100°C for 5 minutes in reducing SDS sample buffer

  • Load equal amounts of protein (typically 20-50 μg) per lane

  • Transfer to PVDF membrane (recommended over nitrocellulose for phospho-proteins)

• For immunohistochemistry/immunofluorescence:

  • Use appropriate fixatives (4% paraformaldehyde is often recommended)

  • For paraffin-embedded tissues, perform antigen retrieval using Tris-EDTA, pH 9.0

  • Block with 5% BSA or serum from the same species as the secondary antibody

• For ELISA:

  • Follow manufacturer's specific fixation and permeabilization protocols

  • For cell-based ELISAs, seed cells at consistent density and treat under identical conditions across wells

• Stimulation conditions:

  • For studying calcium-dependent phosphorylation changes, treat cells with calcium (e.g., 40nM for 30 minutes)

  • For hypoxia-induced changes, use appropriate hypoxic chambers and validated exposure times

Remember that phosphorylation can be very labile, so minimize the time between sample collection and analysis, and avoid repeated freeze-thaw cycles of samples.

How does NFATc3 phosphorylation state affect its interaction with other proteins?

NFATc3 phosphorylation status significantly influences its protein-protein interactions, with important implications for signaling pathways and transcriptional regulation:

• Interaction with GSK-3β: Phosphorylation of NFATc3 enhances its interaction with glycogen synthase kinase 3β (GSK-3β). Research shows that deSUMOylation of NFATc3 at K384 impairs the interaction between NFATc3 and GSK-3β, which subsequently decreases NFATc3 phosphorylation and increases its nuclear occupancy . This suggests a complex interplay between different post-translational modifications in regulating NFATc3 binding partners.

• Interaction with calcineurin: Dephosphorylated NFATc3 has altered binding affinity for the phosphatase calcineurin. Under hypoxia or other stimuli, calcineurin-mediated dephosphorylation is necessary for translocation of deSUMOylated NFATc3 to the nucleus, as demonstrated by experiments with the calcineurin inhibitor Cyclosporin A (CsA) .

• Transcriptional complexes: The phosphorylation state of NFATc3 affects its ability to form functional transcriptional complexes. NFATc3 can function as part of a multicomponent transcription complex consisting of at least two components - a pre-existing cytosolic component and an inducible nuclear component .

• SUMO machinery interaction: Phosphorylation status can influence NFATc3's interaction with the SUMO (Small Ubiquitin-like Modifier) machinery. Under hypoxia, the SUMO protease SENP3 interacts extensively with NFATc3, leading to its deSUMOylation and subsequent altered phosphorylation state .

These complex interaction networks highlight the importance of using phospho-specific tools like Phospho-NFATC3 (Ser165) Antibody to dissect the dynamic regulation of NFATc3 function in different cellular contexts.

What role does NFATc3 Ser165 phosphorylation play in cancer development and progression?

Research using phospho-specific antibodies has revealed critical roles for NFATc3 phosphorylation in cancer:

• Pancreatic ductal adenocarcinoma (PDAC):

  • NFATc3 is constitutively activated in PDAC cells

  • Excessive activation correlates with advanced stages and shorter patient survival

  • Under hypoxic conditions common in tumors, NFATc3 becomes deSUMOylated at K384 by SENP3, which impairs its interaction with GSK-3β, decreases its phosphorylation, and increases nuclear translocation

  • These changes enhance NFATc3-dependent gene expression, promoting tumor cell proliferation, migration, and invasion

• Experimental evidence from tumor models:

  • In xenograft models, PANC-1 cells with intact SENP3 (which promotes NFATc3 dephosphorylation) exhibited rapid tumor growth

  • SENP3 depletion markedly inhibited tumor growth, corresponding with decreased nuclear localization of NFATc3

  • The inhibitory effect of SENP3 depletion was compromised in cells expressing a deSUMOylation-mimetic NFATc3 mutant, confirming the mechanistic link between SUMOylation and phosphorylation in regulating NFATc3 activity in cancer

• Target genes and mechanisms:

  • Phosphorylation-regulated NFATc3 controls expression of genes like MYC that promote cancer progression

  • NFATc3 also regulates release of matrix metalloproteinases (e.g., MMP2) that facilitate invasion and metastasis

These findings highlight the therapeutic potential of targeting NFATc3 phosphorylation pathways in cancer treatment, making phospho-specific antibodies valuable tools for both basic research and drug development efforts.

How can I design experiments to study the interplay between different post-translational modifications of NFATc3?

Investigating the complex interplay between phosphorylation, SUMOylation, and other modifications of NFATc3 requires sophisticated experimental designs:

• Sequential immunoprecipitation approach:

  • First immunoprecipitate with Phospho-NFATC3 (Ser165) Antibody

  • Then probe the immunoprecipitated material with antibodies against other modifications (e.g., SUMO, ubiquitin)

  • Alternatively, perform the immunoprecipitation with anti-SUMO antibodies and then probe with Phospho-NFATC3 (Ser165) Antibody

  • This reveals populations with multiple modifications simultaneously

• Site-directed mutagenesis studies:

  • Generate phospho-mimetic (S165D/E) and phospho-deficient (S165A) mutants

  • Combine with SUMOylation site mutations (e.g., K384R as used in published studies )

  • Analyze the effects on subcellular localization, protein interactions, and transcriptional activity

  • This approach can establish causality and hierarchy between modifications

• Time-course experiments:

  • Stimulate cells with activators like calcium or hypoxia

  • Collect samples at multiple time points (e.g., 0, 5, 15, 30, 60 minutes)

  • Analyze phosphorylation and SUMOylation status simultaneously

  • This reveals the temporal sequence of modifications

• Pharmacological and genetic interventions:

  • Use specific inhibitors of relevant enzymes:

    • Calcineurin inhibitors (e.g., Cyclosporin A) to prevent dephosphorylation

    • GSK-3β inhibitors to prevent phosphorylation

    • SUMOylation inhibitors or SENP3 knockdown/overexpression

  • Monitor changes in all modifications simultaneously

• Advanced microscopy techniques:

  • Perform proximity ligation assays (PLA) to detect closely associated modifications

  • Use fluorescence resonance energy transfer (FRET) with antibodies or tagged constructs

  • Apply live-cell imaging with phospho-sensors to track dynamic changes

These approaches should be combined with functional readouts such as reporter gene assays, ChIP analyses of NFATc3 target genes, and phenotypic assays relevant to the biological process being studied.

What methodological considerations should be addressed when studying NFATc3 phosphorylation dynamics in response to calcium signaling?

Studying calcium-dependent phosphorylation dynamics of NFATc3 requires careful experimental design:

• Calcium stimulation protocols:

  • Use established methods for intracellular calcium elevation:

    • Ionophores (A23187, ionomycin)

    • Thapsigargin (SERCA inhibitor)

    • Physiological agonists (depending on cell type)

  • Published protocols show effectiveness of 40nM calcium for 30 minutes in HeLa cells

  • Consider both the amplitude and duration of calcium signals

• Temporal resolution:

  • NFATc3 shows rapid dephosphorylation and nuclear translocation (complete within 20 minutes)

  • Design time-course experiments with appropriate early time points (e.g., 0, 2, 5, 10, 20, 30 minutes)

  • Use rapid fixation methods to capture transient states

• Phosphorylation site analysis:

  • Ser165 is one of multiple phosphorylation sites on NFATc3

  • Consider using antibodies against other phosphorylation sites for comprehensive analysis

  • Phospho-mass spectrometry can identify all modified sites simultaneously

• Imaging approaches:

  • Combine calcium imaging (Fura-2, Fluo-4, GCaMP) with fixed-time immunofluorescence

  • For live imaging, use fluorescently-tagged NFATc3 constructs with separate calcium indicators

  • Quantify nuclear/cytoplasmic ratios as a measure of translocation

• Phosphatase/kinase involvement:

  • Use calcineurin inhibitors (Cyclosporin A, FK506) to block dephosphorylation

  • Target specific kinases (e.g., GSK-3β knockdown significantly increases depolarization-induced nuclear localization of NFATc family members)

  • Distinguish between maintenance and export phosphorylation events

• Data normalization strategies:

  • For cell-based phosphorylation ELISAs, normalize using one of these approaches:

    • Anti-NFATc3 (total) antibody normalization

    • GAPDH normalization

    • Crystal violet staining normalization

  • For Western blots, normalize phospho-signal to total NFATc3 levels

These methodological considerations ensure robust, reproducible results when investigating the dynamic regulation of NFATc3 phosphorylation in calcium signaling research.

How can I utilize Phospho-NFATC3 (Ser165) Antibody in translational research connecting basic mechanisms to disease states?

Leveraging Phospho-NFATC3 (Ser165) Antibody for translational research requires systematic approaches connecting molecular mechanisms to clinical applications:

• Patient-derived sample analysis:

  • Apply immunohistochemistry with Phospho-NFATC3 (Ser165) Antibody to tissue microarrays from patient cohorts

  • Correlate phosphorylation levels with clinical parameters (disease stage, treatment response, survival)

  • Perform multiplexed immunostaining to simultaneously detect NFATc3 phosphorylation and other markers

  • Published findings show correlation between NFATc3 activation and advanced stages of PDAC with shorter patient survival

• Therapeutic target validation:

  • Screen compound libraries for molecules that modulate NFATc3 Ser165 phosphorylation

  • Use Phospho-NFATC3 (Ser165) Antibody in cell-based phosphorylation ELISAs as a high-throughput screening tool

  • Validate hits with orthogonal assays (Western blot, reporter gene assays)

  • Test promising compounds in disease-relevant models

• Biomarker development:

  • Develop quantitative assays using Phospho-NFATC3 (Ser165) Antibody for clinical samples

  • Optimize protocols for various sample types (tissue, circulating tumor cells, liquid biopsies)

  • Evaluate sensitivity and specificity as a diagnostic or prognostic biomarker

  • Correlate with other established biomarkers

• Disease model characterization:

  • Apply Phospho-NFATC3 (Ser165) Antibody to analyze NFATc3 activity in:

    • Cell line panels representing disease progression

    • Patient-derived xenografts

    • Genetically engineered mouse models

    • 3D organoid cultures

  • Connect phosphorylation status to functional outcomes relevant to disease

• Pathway-targeted approaches:

  • Design combination strategies targeting multiple nodes in the NFATc3 pathway

  • Investigate synergy between modulating NFATc3 phosphorylation and other therapeutic approaches

  • Study resistance mechanisms that emerge through altered phosphorylation patterns

  • Example: Combined targeting of SUMOylation (via SENP3) and phosphorylation pathways in pancreatic cancer

By systematically applying these approaches, researchers can bridge fundamental phosphorylation biology to clinical applications, potentially leading to new diagnostic and therapeutic strategies for diseases involving NFATc3 dysregulation.