Phospho-GATA3 (S308) Recombinant Monoclonal Antibody

What is the functional significance of GATA3 phosphorylation at Serine 308?

Phosphorylation of GATA3 at Serine 308 serves as a key regulatory mechanism affecting its transcriptional activity and protein stability. This post-translational modification has significant implications for T-cell development and differentiation pathways. GATA3 phosphorylation at S308 has been established as a marker of proteasomal turnover specifically in ER-positive breast cancer cells . This phosphorylation event alters GATA3 activity, influencing its role in coordinating macrophage transcriptional activation and UCP2-dependent metabolic reprogramming in response to IL33 signaling . Furthermore, phosphorylation at this residue appears to modulate GATA3's function in driving the differentiation of inflammation-resolving alternatively activated macrophages following tissue injury .

In which research applications can Phospho-GATA3 (S308) antibodies be utilized?

Phospho-GATA3 (S308) recombinant monoclonal antibodies demonstrate versatility across multiple experimental applications:

These applications allow researchers to investigate GATA3 phosphorylation in various experimental contexts, from protein expression analysis to cellular localization studies .

What species reactivity has been confirmed for these antibodies?

Phospho-GATA3 (S308) antibodies have been validated for reactivity with multiple species:

• Human samples: Extensively tested and confirmed

• Mouse samples: Confirmed reactivity

• Rat samples: Confirmed reactivity

This multi-species reactivity makes these antibodies valuable for comparative studies across experimental models, though researchers should verify reactivity when using new sample types or experimental conditions .

How should researchers optimize Western blot conditions for Phospho-GATA3 (S308) detection?

For optimal Western blot results with Phospho-GATA3 (S308) antibodies, implement the following protocol adjustments:

• Sample preparation: Use phosphatase inhibitors in lysis buffers to preserve phosphorylation status

• Blocking conditions: Employ 5% non-fat dry milk (NFDM) in TBST as recommended blocking buffer

• Antibody dilution: Start with 1:1000 dilution for initial optimization, then adjust based on signal intensity (recommended range 1:500-1:10000)

• Positive controls: Include samples known to express phosphorylated GATA3, such as Jurkat cells treated with 8-Bromo-cAMP

• Loading controls: Use appropriate housekeeping proteins that won't interfere with phosphorylated protein detection

These optimization steps ensure specific detection of phosphorylated GATA3 while minimizing background signal and false positives .

What are the critical storage conditions for maintaining antibody performance?

Proper storage is essential for preserving antibody functionality:

• Short-term storage (up to 2 weeks): Store at 4°C

• Long-term storage: Aliquot and store at -20°C

• Avoid freeze-thaw cycles to prevent antibody degradation

• For carrier-free formulations (BSA and azide-free), additional stabilizing agents may be necessary for prolonged storage

Following these storage recommendations ensures antibody integrity and consistent experimental performance over time .

How can Phospho-GATA3 (S308) antibodies be utilized to investigate T-helper cell differentiation mechanisms?

GATA3 is essential for T-helper 2 (Th2) cell differentiation, and its phosphorylation status affects this process. Researchers can employ these advanced methodologies:

• Time-course experiments: Monitor phosphorylation changes during T-cell activation using flow cytometry and Western blotting

• Cytokine stimulation studies: Examine how IL-4, IL-33, and other Th2-promoting cytokines affect GATA3 phosphorylation

• Knockin/knockout approach: Compare wild-type GATA3 with phospho-mutants (S308A) to assess functional consequences

• ChIP-seq analysis: Determine how S308 phosphorylation affects GATA3 binding to target genes using the antibody for chromatin immunoprecipitation

• Single-cell analysis: Combine phospho-GATA3 detection with other markers to identify cellular subpopulations

These approaches can reveal how GATA3 phosphorylation contributes to T-cell lineage commitment and function in immune responses .

What mechanisms regulate GATA3 phosphorylation at S308, and how can they be experimentally investigated?

GATA3 phosphorylation at S308 is regulated through multiple mechanisms:

For comprehensive analysis, researchers should combine these approaches with time-course studies and quantitative Western blotting or ELISA to measure phosphorylation dynamics. Additionally, mass spectrometry can identify other phosphorylation sites that may cross-talk with S308 .

How does GATA3 phosphorylation at S308 contribute to breast cancer biology?

In ER-positive breast cancer cells, GATA3 phosphorylation at S308 functions as a marker of proteasomal turnover . This phosphorylation event has significant implications for breast cancer biology:

• Correlation with tumor progression: Immunohistochemistry using phospho-specific antibodies can reveal associations between phosphorylation status and clinical outcomes

• Estrogen signaling: Experiments combining estrogen treatment with phospho-GATA3 detection can elucidate regulatory mechanisms

• Therapeutic response: Monitoring phospho-GATA3 levels during treatment with proteasome inhibitors or hormone therapies may predict response

• Transcriptional programming: RNA-seq analysis following modulation of GATA3 phosphorylation can identify downstream gene targets

• Metastatic potential: Investigation of phospho-GATA3 in primary versus metastatic samples may reveal its role in disease progression

These research approaches can determine whether targeting GATA3 phosphorylation might offer therapeutic opportunities in breast cancer treatment .

What are the common pitfalls in phospho-specific antibody experiments, and how can they be mitigated?

When working with Phospho-GATA3 (S308) antibodies, researchers should be aware of these potential issues:

• Cross-reactivity concerns: The immunogen sequence for this antibody is identical to the sequence around S340 of GATA2, although phosphorylation at this site has not been reported . Include appropriate negative controls to ensure specificity.

• Phosphorylation preservation: Phosphorylation can be lost during sample preparation. Always use fresh phosphatase inhibitors in lysis buffers and handle samples at 4°C.

• Antibody specificity verification: Validate specificity using:

  • Phosphatase treatment controls

  • Peptide competition assays

  • Phospho-null mutants (S308A)

• Signal optimization: For weak signals:

  • Increase antibody concentration gradually

  • Extend incubation time

  • Use signal enhancement systems like biotin-streptavidin

• Background reduction: If high background occurs:

  • Try alternative blocking buffers (5% BSA instead of milk)

  • Increase washing time and volume

  • Reduce primary antibody concentration

These approaches ensure reliable, reproducible results when investigating GATA3 phosphorylation .

How can researchers quantitatively assess GATA3 phosphorylation levels?

For quantitative analysis of GATA3 phosphorylation:

• Western blot quantification:

  • Use total GATA3 antibody in parallel for normalization

  • Apply densitometry analysis with appropriate software

  • Include standard curves with recombinant phosphorylated protein

• Flow cytometry approach:

  • Perform dual staining with total and phospho-specific antibodies

  • Calculate phosphorylation index as ratio of phospho/total signal

  • Include appropriate isotype controls

• ELISA-based methods:

  • Develop sandwich ELISA with capture/detection antibody pairs

  • Create standard curves using recombinant proteins

  • Calculate phosphorylation ratios across experimental conditions

• Mass spectrometry:

  • Perform immunoprecipitation with total GATA3 antibody

  • Analyze phosphopeptides by LC-MS/MS

  • Quantify phosphorylation stoichiometry

These quantitative approaches provide robust data for comparing GATA3 phosphorylation across experimental conditions .

How might single-cell analysis techniques be applied to investigate heterogeneity in GATA3 phosphorylation?

Single-cell technologies offer powerful approaches for examining GATA3 phosphorylation heterogeneity:

• Single-cell phospho-flow cytometry:

  • Combine phospho-GATA3 (S308) detection with lineage markers

  • Identify cell subpopulations with distinct phosphorylation states

  • Correlate with functional outcomes

• Mass cytometry (CyTOF):

  • Develop metal-conjugated phospho-GATA3 antibodies

  • Simultaneously measure multiple phosphorylation sites

  • Create high-dimensional datasets to identify cellular states

• Imaging mass cytometry:

  • Visualize spatial distribution of phosphorylated GATA3 in tissues

  • Correlate with microenvironmental factors

• Single-cell RNA-seq integration:

  • Combine phospho-protein detection with transcriptomic analysis

  • Identify gene expression signatures associated with phosphorylation states

These approaches can reveal how heterogeneous GATA3 phosphorylation contributes to cellular diversity in immune responses and cancer .

What is the relationship between GATA3 phosphorylation at S308 and its role in macrophage polarization?

GATA3 coordinates macrophage transcriptional activation and metabolic reprogramming in response to IL33, with phosphorylation potentially modulating this activity . To investigate this relationship:

• Macrophage differentiation models:

  • Monitor phospho-GATA3 levels during M1/M2 polarization

  • Compare phosphorylation in tissue-resident versus inflammatory macrophages

  • Assess how IL-33 signaling affects phosphorylation kinetics

• Functional consequences:

  • Express phospho-mimetic (S308D) or phospho-null (S308A) GATA3 mutants

  • Measure impact on UCP2 expression and metabolic parameters

  • Assess effects on anti-inflammatory cytokine production

• In vivo relevance:

  • Examine phospho-GATA3 in tissue injury and resolution models

  • Correlate with macrophage phenotypic markers

  • Assess impact of phosphorylation inhibitors on inflammation resolution

These investigations could reveal how GATA3 phosphorylation serves as a molecular switch in macrophage functional programming during inflammation and tissue repair .

How does phosphorylation at S308 compare with other GATA3 post-translational modifications?

GATA3 undergoes multiple post-translational modifications that collectively regulate its function:

Researchers can investigate these relationships through:

• Sequential immunoprecipitation with modification-specific antibodies

• Mass spectrometry to identify modification patterns

• Mutational studies examining how one modification affects others

This integrated approach provides a comprehensive understanding of GATA3 regulation in different cellular contexts .

How can computational approaches be integrated with phospho-GATA3 antibody data?

Computational methods enhance the value of experimental phospho-GATA3 data:

• Structural modeling:

  • Predict how S308 phosphorylation affects protein conformation

  • Model interactions with DNA and protein partners

  • Simulate dynamic changes in protein structure

• Systems biology approaches:

  • Integrate phosphorylation data into signaling network models

  • Predict downstream effects using pathway analysis

  • Model temporal dynamics of phosphorylation/dephosphorylation

• Machine learning applications:

  • Develop pattern recognition algorithms for image analysis

  • Create predictive models for phosphorylation based on cellular context

  • Identify biomarker signatures incorporating phospho-GATA3 data

• Multi-omics integration:

  • Correlate phosphorylation status with transcriptomic, proteomic, and metabolomic data

  • Construct regulatory networks with phospho-GATA3 as a node

These computational strategies extend the utility of experimental data and generate hypotheses for further investigation .

What are the technical differences between available Phospho-GATA3 (S308) antibody clones?

Several manufacturers produce Phospho-GATA3 (S308) antibodies with distinct characteristics:

When selecting between these options, researchers should consider:

• The specific application requirements

• Whether conjugation to reporters is needed

• The experimental model system

• The degree of validation required for their research

Each antibody may perform differently in specific applications, so preliminary validation is recommended .

How can researchers validate antibody specificity for phosphorylated versus non-phosphorylated GATA3?

Thorough validation of phospho-specificity is crucial:

• Peptide competition assays:

  • Pre-incubate antibody with phosphorylated and non-phosphorylated peptides

  • Only phospho-peptides should block specific binding

• Phosphatase treatment:

  • Treat one sample set with lambda phosphatase

  • Signal should disappear in treated samples

• Genetic validation:

  • Express wild-type GATA3 versus S308A mutant

  • No signal should be detected with the S308A mutant

• Stimulus-response validation:

  • Treat cells with stimuli known to induce phosphorylation (e.g., 8-Bromo-cAMP)

  • Signal should increase in treated samples

• Western blot migration patterns:

  • Phosphorylated proteins often show mobility shifts

  • Confirm band identity through multiple approaches

These validation steps ensure experimental results accurately reflect GATA3 phosphorylation status rather than non-specific binding .

How might Phospho-GATA3 (S308) detection be integrated into multiplexed imaging approaches?

Multiplexed imaging technologies offer powerful ways to study GATA3 phosphorylation in spatial context:

• Multiplex immunofluorescence:

  • Combine phospho-GATA3 with lineage markers and other phospho-proteins

  • Use spectral unmixing to separate fluorophores

  • Apply tissue clearing techniques for 3D imaging

• Mass spectrometry imaging:

  • Utilize metal-tagged antibodies for spatial proteomics

  • Achieve subcellular resolution of phosphorylation patterns

  • Correlate with tissue architecture

• Cyclic immunofluorescence:

  • Perform sequential staining/stripping cycles

  • Build high-parameter datasets within the same tissue section

  • Create spatial maps of signaling networks

• In situ proximity ligation assay:

  • Detect phospho-GATA3 interactions with other proteins

  • Visualize subcellular localization of interaction events

  • Quantify interaction frequency across cell populations

These approaches reveal not just whether GATA3 is phosphorylated but also its spatial relationships with other cellular components .

What role might GATA3 phosphorylation play in novel therapeutic approaches?

Understanding GATA3 phosphorylation mechanisms opens new therapeutic possibilities:

• Targeted inhibition approaches:

  • Design small molecules targeting kinases that phosphorylate S308

  • Develop peptide inhibitors that prevent phosphorylation

  • Create proteolysis-targeting chimeras (PROTACs) specific for phospho-GATA3

• Diagnostic applications:

  • Develop phospho-GATA3 detection in liquid biopsies

  • Create companion diagnostics for breast cancer treatments

  • Establish prognostic scoring systems incorporating phosphorylation status

• Immunotherapy connections:

  • Investigate how T-cell GATA3 phosphorylation affects immunotherapy response

  • Explore manipulation of GATA3 phosphorylation to enhance anti-tumor immunity

  • Study macrophage reprogramming through GATA3 phosphorylation modulation

• Cell-based therapeutics:

  • Engineer T-cells with phospho-mimetic or phospho-null GATA3 for enhanced function

  • Modify regulatory T-cells through GATA3 phosphorylation manipulation