Phospho-NFAT5 (Ser155) Antibody

What is the functional significance of NFAT5 Ser155 phosphorylation in cellular responses?

Phosphorylation of NFAT5 at Serine 155 represents a key regulatory modification that influences its transcriptional activity. In research contexts, this phosphorylation event is associated with:

• Enhanced NFAT5-mediated gene activation during hypertonic stress

• Modulation of nuclear localization and DNA binding capability

• Regulation of NFAT5's role in amplifying TLR-induced antipathogen transcription responses

NFAT5 plays a pivotal role in facilitating chromatin accessibility, particularly at promoter regions of multiple TLR4-responsive genes. It induces H3K27me3 demethylation, which serves as an early mechanism that facilitates p65/NF-κB recruitment to promoters of various TLR4-induced genes .

How should researchers validate the specificity of Phospho-NFAT5 (Ser155) antibodies?

Proper validation of phospho-specific antibodies is critical for experimental reliability. For Phospho-NFAT5 (Ser155) antibodies, the following validation protocol is recommended:

• Phosphatase treatment control: Treat cell lysates with lambda phosphatase to remove phosphorylation and confirm loss of signal

• Stimulation-dependent detection: Compare unstimulated versus hypertonic stress-stimulated cells (300-500 mOsm)

• Peptide competition assay: Pre-incubate antibody with phosphorylated and non-phosphorylated peptides

• Knockout/knockdown control: Include NFAT5-deficient cells as negative controls

The antibody should specifically detect NFAT5 only when phosphorylated at Ser155, showing decreased signal in phosphatase-treated samples and NFAT5-deficient cells .

What are the optimal experimental conditions for detecting NFAT5 Ser155 phosphorylation in macrophages?

When studying NFAT5 phosphorylation in macrophage responses, researchers should consider these methodological approaches:

• Stimulation parameters:

  • Low-dose TLR4 stimulation: 0.05-0.1 ng/ml LPS (critical for observing NFAT5-dependent effects)

  • Hypertonic conditions: NaCl or sorbitol to increase osmolarity by 50-100 mOsm

  • Timing: Initial phosphorylation typically occurs within 30-60 minutes

• Cell preparation:

  • Primary macrophages can be isolated using anti-CD11b antibodies and magnetic beads

  • Peritoneal macrophages obtained 3 hours after LPS injection (0.05 μg/kg) provide physiologically relevant samples

• Sample processing:

  • Rapid cell lysis in buffers containing phosphatase inhibitors

  • Processing at 4°C to prevent dephosphorylation

  • Recommended dilution for Western blot: 1:500-1:1000

What experimental evidence demonstrates the relationship between NFAT5 phosphorylation and chromatin remodeling?

Advanced research has revealed NFAT5's role in chromatin accessibility, with Ser155 phosphorylation potentially mediating these effects:

• Transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) analysis demonstrates that NFAT5:

  • Facilitates chromatin accessibility primarily at promoter regions of multiple TLR4-responsive genes

  • Functions most critically under conditions of mild TLR stimulation

• Histone modification analysis shows NFAT5-dependent mechanisms:

  • H3K27me3 demethylation as an early NFAT5-dependent process

  • This demethylation facilitates p65/NF-κB recruitment to promoters

  • Inhibition of H3K27me3 demethylases impairs p65/NF-κB recruitment to levels similar to NFAT5-deficient cells

The mechanistic relationship is evident in experiments showing that NFAT5 is required for effective recruitment of central effectors p65/NF-κB and c-Fos to specific proinflammatory target genes including Nos2, Il6, and Tnf in primary macrophages responding to low doses of LPS .

How does NFAT5 function in response to different intensities of TLR stimulation?

NFAT5's role varies depending on stimulation intensity, which is important for experimental design:

This dose-dependent function allows NFAT5 to optimize the expression of genes with different transcription requirements. NFAT5 is readily recruited to the promoters of secondary response genes (Nos2, Il6, Ptgs2) in a TLR-dependent manner but is constitutively bound to promoters of primary response target genes (Tnf, Il1a, Ccl2, Traf1) in steady-state macrophages .

What is the proper methodology for using Phospho-NFAT5 (Ser155) antibodies in Western blotting?

For optimal Western blot results with Phospho-NFAT5 (Ser155) antibodies:

• Sample preparation:

  • Lyse cells in buffer containing phosphatase inhibitors (Na3VO4, NaF, β-glycerophosphate)

  • Maintain cold conditions throughout processing

  • Use fresh samples when possible or store at -80°C with protease/phosphatase inhibitors

• Electrophoresis conditions:

  • Use low percentage gels (6-8%) due to NFAT5's high molecular weight (~210 kDa)

  • Include positive controls (cells treated with hypertonic conditions)

• Antibody application:

  • Recommended dilution: 1:500-1:2000 for Western blotting

  • Overnight incubation at 4°C for primary antibody

  • Block with 5% BSA (not milk) to prevent interference with phospho-epitopes

• Signal detection:

  • Enhanced chemiluminescence with extended exposure times may be necessary

  • Use high-sensitivity detection systems for low abundance phosphorylated protein

How can researchers distinguish between NFAT5 phosphorylation at Ser155 versus other phosphorylation sites?

Multiple phosphorylation sites exist on NFAT5, requiring careful experimental design:

• Comparative analysis:

  • Use antibodies specific to different phosphorylation sites (e.g., Ser155 vs Ser145)

  • Compare phosphorylation kinetics under various stimulation conditions

  • Analyze using phospho-site mutants (S155A) to confirm specificity

• Mass spectrometry approach:

  • Immunoprecipitate NFAT5 and analyze by phospho-peptide mapping

  • Compare phosphorylation profiles under different conditions

  • Quantify relative abundance of phosphorylation at different sites

• Functional correlation:

  • Compare biological outcomes of mutations at different phosphorylation sites

  • Assess transcriptional activity using reporter assays with phospho-mimetic mutants

Studies have shown that phosphorylation at Ser155 has distinct functional consequences compared to other sites, making site-specific antibodies essential for mechanistic studies.

What are the considerations when using Phospho-NFAT5 (Ser155) antibodies for studying pathogen responses?

When investigating NFAT5's role in antipathogen responses:

• Experimental timing:

  • Early time points (1-3 hours) for phosphorylation events

  • Later time points (6-24 hours) for gene expression analysis

• Pathogen selection:

  • TLR4 agonists like LPS are well-characterized for NFAT5 activation

  • NFAT5's role in different TLR pathways (TLR2, TLR3, etc.) requires specific optimization

  • Leishmania infection models have shown NFAT5-dependent control

• Readout systems:

  • Measure target gene expression (iNOS, IL-6, TNF-α, CCL2)

  • Assess functional outcomes (NO production, bacterial killing)

  • Monitor chromatin accessibility and histone modifications

• Controls:

  • Include NFAT5-deficient macrophages (LysM-Cre or Csf1r-Cre crossed with NFAT5-floxed mice)

  • Compare low vs. high pathogen burden conditions

How does NFAT5 phosphorylation contribute to biomolecular condensate formation?

Recent advanced research has revealed that NFAT5 forms dynamic, reversible biomolecular condensates in response to increasing ionic strength:

• Condensate formation mechanism:

  • NFAT5 directly senses solution ionic strength using its C-terminal intrinsically disordered region

  • This self-associative property is conserved from insects to mammals

  • Phosphorylation status, including at Ser155, may regulate condensation properties

• Functional significance:

  • Condensate formation allows NFAT5 to accumulate in the nucleus

  • This accumulation activates genes that restore cellular ion content

  • Both reduced condensation and excessive aggregation impair NFAT5 activity

• Experimental approaches to study condensates:

  • Fluorescence recovery after photobleaching (FRAP) to assess dynamics

  • Phase separation assays with purified components

  • Live cell imaging with fluorescently tagged NFAT5

  • Mutation analysis to identify regions critical for condensation

Research has demonstrated that human NFAT5 alone is sufficient to reconstitute a mammalian transcriptional response to ionic or hypertonic stress in yeast, indicating its autonomous sensing capability .

What methodological approaches can detect the interaction between phosphorylated NFAT5 and chromatin?

To investigate how phosphorylated NFAT5 interacts with chromatin:

• Chromatin immunoprecipitation (ChIP):

  • Use Phospho-NFAT5 (Ser155) antibodies for direct ChIP

  • Sequence analysis of bound regions (ChIP-seq)

  • Focus on promoters of known target genes (Nos2, Il6, Tnf, Akr1b3, Slc5a3, Slc6a12)

• Transposase-accessible chromatin analysis:

  • ATAC-seq to map regions where NFAT5 induces accessibility changes

  • Compare wild-type vs. NFAT5-deficient cells

  • Analyze under various stimulation conditions

• Histone modification mapping:

  • ChIP-seq for histone marks (H3K27me3, H3K4me3, etc.)

  • Correlation with NFAT5 binding and phosphorylation status

  • Inhibitor studies of chromatin-modifying enzymes

Research has shown that NFAT5 facilitates chromatin accessibility specifically at promoter regions and enhances H3K27me3 demethylation, which is critical for subsequent recruitment of transcription factors like p65/NF-κB .

How should researchers interpret discrepancies in NFAT5 phosphorylation results across different experimental models?

When facing conflicting results across models:

• Source variations:

  • Cell type differences: Primary macrophages vs. cell lines vs. in vivo models

  • Species differences: Human vs. mouse (despite high conservation)

  • Genetic background effects: Pure vs. mixed background mice

• Technical considerations:

  • Antibody specificity: Different commercial antibodies may have varying sensitivities

  • Phosphorylation stability: Rapid dephosphorylation during sample processing

  • Detection methods: Western blot vs. flow cytometry vs. immunofluorescence

• Stimulus differences:

  • Nature of stimulus: Ionic vs. non-ionic osmolytes

  • Intensity of stimulus: Low vs. high TLR activation

  • Duration of stimulus: Acute vs. chronic exposure

• Resolution approaches:

  • Use multiple antibodies and detection methods

  • Include appropriate positive and negative controls

  • Validate with genetic approaches (phospho-mimetic or phospho-dead mutants)

What is known about the role of NFAT5 Ser155 phosphorylation in disease contexts?

Emerging research connects NFAT5 phosphorylation to various pathological conditions:

• Immune dysregulation:

  • NFAT5 haploinsufficiency has been reported in two patients with Epstein-Barr virus susceptibility

  • One case involved chronic-active infection of the liver and bowel

  • Another case resulted in fatal hemophagocytic lymphohistiocytosis

• Kidney disease:

  • NFAT5 is critical in the renal medulla where high interstitial osmolarity allows water reabsorption

  • Emerging evidence connects NFAT5 dysfunction to chronic kidney disease of unknown origin

  • This may result from failing adaptive mechanisms against extreme or prolonged hypertonic stress

• Climate-related diseases:

  • Rising temperatures and scarcity of potable water contribute to conditions affecting tissues vulnerable to hypertonic stress

  • NFAT5's phosphorylation status, including at Ser155, may influence adaptive responses

  • This has implications for emerging public health challenges in changing climate conditions