Phospho-NFATC4 (S676) Antibody

What is the NFAT family and what cellular functions does NFATc4 regulate?

The Nuclear Factor of Activated T-cells (NFAT) family comprises calcium-regulated transcription factors involved in multiple physiological processes. NFATc4 specifically functions in immune regulation, cardiovascular development, musculoskeletal function, and nervous system development . At the molecular level, NFATc4 controls the transcription of cytokine genes including IL-2 and IL-4, and partners with various transcription regulators including GATA (for heart development), FOXP3 (immune tolerance regulation), AP-1 (T-cell response), and MEF (muscle development) . In metabolic contexts, NFATc4 plays roles in adipocyte differentiation and glucose/insulin homeostasis . The ability of NFATc4 to respond to calcium/calcineurin signaling makes it a versatile regulator of multiple developmental and cellular processes.

What does Phospho-NFATc4 (S676) Antibody specifically detect?

Phospho-NFATc4 (S676) Antibody detects endogenous levels of the NFATc4 protein exclusively when phosphorylated at the serine 676 residue . This specificity is crucial for studying the phosphorylation-dependent regulation of NFATc4. The antibody is engineered to recognize a specific synthesized peptide derived from human NFAT3 (another name for NFATc4) surrounding the phosphorylation site of Ser676, typically covering amino acids 642-691 . This highly specific detection capability allows researchers to distinguish between phosphorylated and non-phosphorylated forms of NFATc4, providing insight into its activation state under various experimental conditions.

What applications is Phospho-NFATc4 (S676) Antibody suitable for?

Phospho-NFATc4 (S676) Antibody is suitable for multiple experimental applications, including Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), and Enzyme-Linked Immunosorbent Assay (ELISA) . For Western Blot applications, the recommended dilution range is 1:500-1:2000 . For Immunohistochemistry, researchers should use dilutions of 1:100-1:300 . For Immunofluorescence applications, dilutions of 1:50-200 are recommended . For ELISA, higher dilutions of 1:5000 are typically used . The versatility across multiple immunological techniques makes this antibody valuable for comprehensive experimental designs investigating NFATc4 phosphorylation in different contexts.

How does NFATc4 phosphorylation status affect its localization and function?

NFATc4 phosphorylation status critically determines its subcellular localization and transcriptional activity. The protein exists in a highly phosphorylated state in the cytosol under basal conditions . Upon elevation of intracellular calcium levels, calcineurin (a calcium-dependent phosphatase) dephosphorylates NFATc4, triggering its translocation from the cytoplasm to the nucleus where it can activate target gene transcription .

Specifically, NFATc4 can be phosphorylated at multiple sites by different kinases: MTOR, IRAK1, MAPK7/ERK5, and MAPK14/p38 phosphorylate Ser-168 and Ser-170; MAPK8/JNK1 and MAPK9/JNK2 phosphorylate Ser-213 and Ser-217; and RPS6KA3 phosphorylates Ser-289 and Ser-344 . Phosphorylation at Ser676 represents an important regulatory site for NFATc4 activity. Notably, phosphorylation by GSK3B markedly increases NFATc4 ubiquitination, leading to degradation by the proteasome . UV irradiation stimulates phosphorylation at Ser-168 and Ser-170 . MAPK7/ERK5 and MTOR specifically regulate NFATc4 nuclear export through phosphorylation at Ser-168 and Ser-170 .

What role does NFATc4 phosphorylation play in inflammation and disease models?

NFATc4 phosphorylation status regulates inflammatory processes across multiple disease contexts. In cholestatic liver disease, NFAT signaling regulates the expression of inflammatory genes . Research has demonstrated that inhibitors of the Ca²⁺/calcineurin/NFAT signaling pathway significantly repress taurocholic acid (TCA) induction of inflammatory cytokines in mouse hepatocytes .

In rheumatoid arthritis (RA), although the exact role of specific NFAT isoforms remains to be fully elucidated, nuclear localization of NFAT proteins in synovial cells of RA joints indicates their activity in disease progression . Future quantitative examination of individual NFAT isoform activity (including phosphorylated NFATc4) in macrophages of synovial fluid from RA joints would provide valuable insights into their roles in joint diseases .

In kidney diseases such as idiopathic focal segmental glomerulosclerosis (FSGS), treatment with inhibitors of the calcineurin-NFAT pathway (cyclosporine A, FK506) ameliorates proteinuria and improves symptoms, suggesting NFAT's involvement in disease pathogenesis . Monitoring NFATc4 phosphorylation status could provide biomarkers for disease progression and treatment response.

How can Phospho-NFATc4 (S676) Antibody be used in studying cardiac and neural development?

For cardiac research, Phospho-NFATc4 (S676) Antibody can be utilized to investigate NFATc4's role in embryonic heart development, where it works in conjunction with NFATc3 . NFATc4 transactivates many genes crucial for cardiovascular system function, including AGTR2, NPPB/BNP (in synergy with GATA4), NPPA/ANP/ANF, and MYH7/beta-MHC . Experimental designs could incorporate this antibody to monitor how phosphorylation at Ser676 affects NFATc4's interaction with NFATC3 and STAT3 in activating cardiac-specific gene promoters, such as the alpha-beta E4 promoter region of CRYAB in cardiomyocytes .

For neuroscience applications, the antibody can help elucidate NFATc4's functions in adult hippocampal neurogenesis, BDNF-driven pro-survival signaling in hippocampal adult-born neurons, and formation of long-term spatial memory and long-term potentiation . In cochlear nucleus neurons, NFATc4 may influence deafferentation-induced apoptosis during the developmental critical period . Researchers could design experiments using this antibody to track NFATc4 phosphorylation status during critical developmental windows or in response to neuronal stimulation.

What are the optimal protocols for using Phospho-NFATc4 (S676) Antibody in Western blotting?

For optimal Western blot results with Phospho-NFATc4 (S676) Antibody, follow these methodological guidelines:

• Sample preparation: Treat cells with appropriate stimuli that affect NFATc4 phosphorylation status. For example, treating HepG2 cells with Ca²⁺ (40μM for 30 minutes) has been demonstrated to affect NFATc4 phosphorylation .

• Lysate preparation: Prepare cell lysates in an appropriate lysis buffer containing protease and phosphatase inhibitors to preserve phosphorylation status. IP-lysis buffer (50 mM HEPES pH 7.3, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing protease inhibitors (PMSF, TPCL, and TLCK) has been used successfully in NFAT research .

• Protein quantification: Determine protein concentration and load equal amounts (typically 20-40 μg) per lane.

• Electrophoresis and transfer: Separate proteins by SDS-PAGE and transfer to a PVDF or nitrocellulose membrane.

• Blocking: Block the membrane in 5% BSA in TBST (preferred over milk for phospho-specific antibodies).

• Primary antibody incubation: Dilute Phospho-NFATc4 (S676) Antibody at 1:500-1:2000 in blocking buffer and incubate overnight at 4°C .

• Controls: Include a phospho-peptide blocking control, as demonstrated in Western blot analysis of lysates from HepG2 cells, where the right lane was blocked with the phospho peptide to confirm specificity .

• Visualization: Use appropriate secondary antibodies and detection systems suited to your laboratory setup.

• Storage: Store antibody at -20°C for up to 1 year and avoid repeated freeze-thaw cycles to maintain activity .

What are the recommended procedures for immunohistochemistry with Phospho-NFATc4 (S676) Antibody?

For effective immunohistochemistry using Phospho-NFATc4 (S676) Antibody, follow these methodological recommendations:

• Tissue preparation: Use formalin-fixed, paraffin-embedded tissue sections (human tonsil has been successfully used as demonstrated in the literature) .

• Antigen retrieval: Perform antigen retrieval using Tris-EDTA buffer at pH 9.0, as this has been validated for optimal results with this antibody .

• Blocking: Block endogenous peroxidase activity and non-specific binding sites with appropriate blocking reagents.

• Primary antibody application: Dilute the Phospho-NFATc4 (S676) Antibody at 1:100-1:300 and incubate overnight at 4°C .

• Secondary antibody: Apply an appropriate species-specific secondary antibody at 1:200 dilution and incubate at room temperature for approximately 30 minutes .

• Detection and counterstaining: Use a suitable detection system followed by hematoxylin counterstaining, dehydration, and mounting.

• Controls: Include positive control tissues where NFATc4 phosphorylation is known to occur, and negative controls (primary antibody omitted) to validate specificity.

How should researchers design experiments to study NFATc4 phosphorylation in different cellular contexts?

When designing experiments to study NFATc4 phosphorylation across different cellular contexts, consider these methodological approaches:

• Cell/tissue selection: Choose appropriate cell types or tissues where NFATc4 is expressed and functionally relevant. These may include:

  • Immune cells (T cells) for studying immune response

  • Cardiomyocytes for cardiovascular research

  • Hepatocytes for liver inflammation studies

  • Neurons for neurological investigations

  • Adipocytes for metabolic research

• Stimulation conditions:

  • For calcium-dependent activation: Use calcium ionophores (like ionomycin) or physiological stimuli that elevate intracellular calcium

  • For inflammatory contexts: Treat with cytokines (TNFα, IL-1β) or other inflammatory stimuli

  • For metabolic studies: Consider glucose or insulin treatments

  • For specific pathway activation: Employ pathway-specific activators or inhibitors

• Time course studies: Establish the temporal dynamics of NFATc4 phosphorylation by collecting samples at multiple time points after stimulation.

• Inhibitor studies: Use specific inhibitors of:

  • Calcineurin (cyclosporine A, FK506) to prevent NFAT dephosphorylation

  • Kinases that phosphorylate NFATc4 (GSK3B, MAPK7/ERK5, MTOR, etc.)

• Subcellular fractionation: Separate nuclear and cytoplasmic fractions to track NFATc4 translocation in response to phosphorylation changes.

• Co-immunoprecipitation: Identify interaction partners influenced by phosphorylation status using techniques like those described in the literature: immunoprecipitation with 2.5 μg antibody and 50 μl protein A Sepharose beads, overnight incubation at 4°C, followed by washing in NP40 IP-buffer .

• Reporter assays: Implement luciferase reporter constructs containing NFAT-responsive elements to measure transcriptional activity as a function of phosphorylation status .

How can researchers troubleshoot weak or absent signals when using Phospho-NFATc4 (S676) Antibody?

When encountering weak or absent signals with Phospho-NFATc4 (S676) Antibody, consider these methodological solutions:

• Antibody concentration: Adjust the antibody dilution within the recommended range (1:500-1:2000 for WB, 1:100-1:300 for IHC) . For weak signals, try a more concentrated antibody solution.

• Phosphorylation status verification: Ensure your experimental conditions effectively induce NFATc4 phosphorylation at Ser676. Test positive control treatments such as calcium stimulation (40μM Ca²⁺ for 30 minutes has been verified for HepG2 cells) .

• Phosphatase inhibitors: Ensure complete phosphatase inhibitor cocktails are used during sample preparation to preserve phosphorylation status.

• Antigen retrieval optimization: For IHC applications, optimize antigen retrieval conditions. Tris-EDTA at pH 9.0 has been validated, but modifications to treatment duration or temperature may be necessary for specific tissues .

• Blocking optimization: For Western blots, use BSA rather than milk for blocking and antibody dilution, as milk contains phosphatases that might reduce signal for phospho-specific antibodies.

• Detection system sensitivity: Use more sensitive detection systems such as enhanced chemiluminescence (ECL) plus or super signal reagents for Western blots, or amplification systems for IHC.

• Exposure time: For Western blots, increase exposure time when capturing images.

• Protein loading: Increase the amount of total protein loaded to enhance detection of low-abundance phosphorylated species.

What are the critical factors for ensuring specificity when using Phospho-NFATc4 (S676) Antibody?

To ensure specificity when using Phospho-NFATc4 (S676) Antibody, implement these methodological controls and considerations:

• Phospho-peptide competition: Perform a blocking experiment using the specific phospho-peptide antigen against which the antibody was raised, as demonstrated in Western blot analysis where the right lane was blocked with the phospho-peptide .

• Dephosphorylation controls: Treat a portion of your samples with lambda phosphatase prior to analysis to confirm signal is phosphorylation-dependent.

• Stimulation/inhibition pairs: Compare samples from conditions known to increase NFATc4 Ser676 phosphorylation with those treated with specific inhibitors of the responsible kinases.

• Genetic controls: Where possible, use NFATC4 knockout or knockdown samples as negative controls.

• Cross-reactivity assessment: Verify that the antibody doesn't detect other phosphorylated NFAT family members (NFATc1, NFATc2, NFATc3) by testing pure proteins or overexpression systems.

• Antibody validation: Confirm the antibody was affinity-purified using epitope-specific immunogen, as this increases specificity .

• Species considerations: Ensure the antibody is validated for your species of interest. The Phospho-NFATc4 (S676) Antibody has been confirmed to react with human and mouse NFATc4 .

How can the Phospho-NFATc4 (S676) Antibody be integrated into multi-parameter experimental designs?

For integrating Phospho-NFATc4 (S676) Antibody into complex, multi-parameter experiments, consider these methodological approaches:

• Multiplex immunofluorescence: Combine Phospho-NFATc4 (S676) Antibody with antibodies against other signaling proteins (using different species antibodies or directly conjugated fluorophores) to simultaneously assess multiple pathways. Potential partners include:

  • Total NFATc4 (to calculate phosphorylation ratio)

  • Calcineurin components

  • Upstream kinases (MTOR, MAPK7/ERK5, GSK3B)

  • Downstream targets

• Pathway analysis integration: Combine phospho-NFATc4 detection with assessment of:

  • Calcium signaling components

  • Other transcription factors that partner with NFAT (AP-1, GATA4, FOXP3)

  • Inflammatory mediators in disease models

• Sequential immunoprecipitation: First immunoprecipitate with Phospho-NFATc4 (S676) Antibody, then probe for interaction partners to map phosphorylation-dependent protein complexes.

• ChIP-seq applications: Use the antibody for chromatin immunoprecipitation followed by sequencing to identify genome-wide binding sites of phosphorylated NFATc4, as the transcription factor binds the consensus DNA sequence 5'-GGAAAAT-3' .

• Reporter system integration: Incorporate into two-color reporter systems like those used to quantify NFAT and NF-κB signaling levels upon activation, as described in CAR-T cell research .

• Flow cytometry: Adapt for intracellular phospho-flow cytometry to assess NFATc4 phosphorylation at the single-cell level in heterogeneous populations.

• Mass spectrometry coupling: Use the antibody to enrich phosphorylated NFATc4 prior to mass spectrometry analysis to identify novel post-translational modifications or interaction partners.

How can Phospho-NFATc4 (S676) Antibody be used to investigate NFAT signaling in inflammatory liver diseases?

For investigating NFAT signaling in inflammatory liver diseases, researchers can utilize Phospho-NFATc4 (S676) Antibody in these methodological approaches:

• Cholestatic liver disease models: The antibody can be used to track NFATc4 phosphorylation status in both in vivo cholestatic models and primary hepatocyte cultures treated with bile acids such as taurocholic acid (TCA) .

• Phosphorylation dynamics: Monitor changes in NFATc4 Ser676 phosphorylation in response to:

  • Bile acid accumulation

  • Inflammatory cytokine stimulation

  • Treatment with Ca²⁺/calcineurin/NFAT pathway inhibitors

• Subcellular localization studies: Track the nuclear translocation of dephosphorylated NFATc4 in cholestatic hepatocytes using immunofluorescence with Phospho-NFATc4 (S676) Antibody (cytoplasmic signal) in combination with total NFATc4 antibody .

• Gene regulation analysis: Combine with ChIP-PCR to determine NFATc4 binding to promoters of inflammatory genes in hepatocytes under cholestatic conditions .

• Therapeutic evaluation: Assess the effect of potential therapeutic compounds on NFATc4 phosphorylation status and subsequent inflammatory gene expression.

• Patient sample analysis: Apply immunohistochemistry protocols using Phospho-NFATc4 (S676) Antibody on liver biopsy samples from patients with primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) to correlate phosphorylation patterns with disease severity .

What approaches can be used to study NFATc4 phosphorylation in metabolic disorders?

To investigate NFATc4 phosphorylation in metabolic disorders, researchers can implement these methodological strategies using Phospho-NFATc4 (S676) Antibody:

• Adipocyte differentiation models: Monitor NFATc4 phosphorylation changes during adipocyte differentiation, as NFATc4 plays a role in this process .

• Glucose/insulin signaling: Assess how NFATc4 phosphorylation at Ser676 responds to glucose and insulin treatments in relevant cell types, given NFAT's role in glucose and insulin homeostasis .

• Tissue-specific analyses: Compare NFATc4 phosphorylation patterns across metabolically active tissues (adipose, liver, muscle, pancreas) in normal versus disease models.

• Gene regulation studies: Investigate how NFATc4 phosphorylation status affects its binding to:

  • PPARG gene promoter (important in adipocyte function)

  • Resistin promoter (amplified from mouse genomic DNA and subcloned into pGL3-luciferase reporter plasmid using NheI and XhoI restriction sites)

• Interaction with metabolic regulators: Assess how NFATc4 phosphorylation affects its interaction with key metabolic regulators such as PPARγ and C/EBPα .

• Signaling crosstalk: Investigate relationships between NFATc4 and other metabolic signaling components like insulin receptor (IR), Akt, ERK, and S6K .

• Therapeutic implications: Evaluate how anti-diabetic or anti-obesity compounds affect NFATc4 phosphorylation and subsequent metabolic gene regulation.

How can researchers apply Phospho-NFATc4 (S676) Antibody in cancer immunotherapy research?

For applications in cancer immunotherapy research, Phospho-NFATc4 (S676) Antibody can be utilized in these methodological frameworks:

• CAR-T cell engineering: Investigate NFATc4 phosphorylation status in chimeric antigen receptor (CAR) T-cell development, particularly in cells constructed with varied signaling modules from natural co-stimulatory receptors .

• Signaling quantification: Incorporate into two-color reporter systems (like those established in Jurkat cells) to quantify NFAT and NF-κB signaling levels upon CAR activation .

• Modular recombination strategy: Assess how different synthetic co-signaling modules affect NFATc4 phosphorylation patterns in engineered immune cells .

• Comparative analysis: Evaluate NFATc4 phosphorylation across conventional versus enhanced CAR designs (such as αCD19-HVEM-ζ and αCD19-CD27-ζ CAR) to understand signaling differences .

• Libraries of recombinant CARs: Track NFATc4 phosphorylation across libraries of CARs with rationally rewired co-signaling modules derived from co-signaling receptors (CD28, ICOS, 4-1BB, OX40) .

• Correlation with function: Correlate NFATc4 Ser676 phosphorylation levels with:

  • Cytokine production profiles

  • Cytotoxic capacity against tumor targets

  • CAR-T cell persistence

  • Anti-tumor efficacy in preclinical models

• Optimization parameters: Use as a biomarker to optimize CAR design for enhanced efficacy and reduced toxicity in next-generation immunotherapies.