Phospho-GATA4 (Ser105) Antibody

What is GATA4 and what role does phosphorylation at Ser105 play in its function?

GATA4 is a transcription factor belonging to the GATA family of zinc-finger transcription factors that binds to the consensus sequence 5'-AGATAG-3'. It plays a critical role in cardiac development and function, regulating genes involved in embryogenesis and myocardial differentiation. GATA4 has a molecular weight of approximately 44 kDa and functions as a transcriptional activator .

Phosphorylation at Serine 105 is a post-translational modification that regulates GATA4's transcriptional activity. This phosphorylation is mediated primarily by extracellular signal-regulated kinases (ERKs) and enhances GATA4's DNA binding ability and transcriptional activity in cardiac cells. The phosphorylation at Ser105 is particularly important during cardiac hypertrophy and developmental processes, where it increases GATA4's capacity to activate cardiac genes in cooperation with other transcription factors such as NKX2-5 .

How does Phospho-GATA4 (Ser105) differ from other phosphorylation sites on GATA4?

GATA4 contains multiple phosphorylation sites that regulate its activity through different signaling pathways. While Ser105 phosphorylation is primarily mediated by ERK signaling pathways and enhances DNA binding affinity, other sites serve distinct functions:

The specificity of Phospho-GATA4 (Ser105) antibodies is critical because they detect GATA4 only when phosphorylated at Serine 105, allowing researchers to specifically monitor this particular activation state without detecting other phosphorylation events . This specificity enables precise tracking of ERK-mediated activation of GATA4 in experimental contexts.

What are the validated applications for Phospho-GATA4 (Ser105) antibodies in cardiac research?

Phospho-GATA4 (Ser105) antibodies have been validated for multiple applications in cardiac research:

• Western Blotting (WB): For quantitative assessment of phosphorylation levels in cardiac tissue or cultured cardiomyocytes, typically using 1:500-1:2000 dilutions. This application allows researchers to monitor changes in GATA4 phosphorylation in response to hypertrophic stimuli or developmental cues .

• Immunohistochemistry (IHC): For visualizing the spatial distribution of phosphorylated GATA4 in cardiac tissue sections, typically at 1:50-1:200 dilution. This application is valuable for developmental studies and pathological analyses .

• Immunofluorescence (IF): For co-localization studies with other cardiac transcription factors or signaling molecules, using 1:100-1:500 dilution. This technique enables high-resolution analysis of nuclear localization and co-regulatory complexes .

• ELISA: For high-throughput screening of phosphorylation levels in multiple samples, using approximately 1:20000 dilution. This application is useful for drug screening or time-course experiments .

All these applications contribute to understanding GATA4's role in cardiac development, hypertrophy, and heart failure models .

How can I verify the specificity of a Phospho-GATA4 (Ser105) antibody in my experimental system?

Verifying antibody specificity is critical for reliable results. For Phospho-GATA4 (Ser105) antibodies, implement these validation approaches:

• Phosphatase treatment control: Treat half of your protein sample with lambda phosphatase before Western blotting. The signal should disappear in the treated sample if the antibody is phospho-specific .

• Peptide competition assay: Pre-incubate the antibody with the phosphorylated peptide used as the immunogen. This should block specific binding and eliminate the signal, as demonstrated in Western blot analyses of HepG2 cell extracts .

• Phosphorylation induction: Treat cells with agents known to activate the ERK pathway (like phenylephrine for cardiomyocytes) to increase Ser105 phosphorylation, and compare with inhibitor-treated samples (like U0126 for MEK/ERK inhibition) .

• Mutant constructs: Express wild-type GATA4 alongside a S105A mutant (serine to alanine) that cannot be phosphorylated at this site. The antibody should only detect the wild-type protein after stimulation .

• Kinase assays: Perform in vitro kinase assays with recombinant GATA4 and active ERK2, then probe with the phospho-antibody to confirm detection of the newly phosphorylated site .

What are optimal sample preparation methods for detecting phosphorylated GATA4?

Detecting phosphorylated GATA4 requires careful sample preparation to preserve the phosphorylation state:

• Lysis buffer composition: Use a phosphatase inhibitor-enriched lysis buffer containing:

  • 50 mM Tris-HCl (pH 7.4)

  • 150 mM NaCl

  • 1% NP-40 or Triton X-100

  • 1 mM EDTA

  • 10 mM NaF (phosphatase inhibitor)

  • 2 mM Na3VO4 (phosphatase inhibitor)

  • 1 mM PMSF (protease inhibitor)

  • Commercial protease inhibitor cocktail

• Sample handling: Keep samples cold throughout processing (0-4°C) and work quickly to minimize dephosphorylation .

• Tissue processing: For cardiac tissue, rapid freezing in liquid nitrogen immediately after collection is essential. For optimal phosphoprotein preservation, consider perfusing the tissue with phosphatase inhibitors before collection .

• Fixation for microscopy: For IHC/IF applications, 4% paraformaldehyde fixation with phosphatase inhibitors in the fixative solution helps maintain phosphorylation states. Cold methanol fixation can also preserve phospho-epitopes .

• Membrane blocking: For Western blots, BSA is preferable to milk as a blocking agent, as milk contains phosphatases that may reduce signal from phospho-proteins .

How should I troubleshoot weak or non-specific signals when using Phospho-GATA4 (Ser105) antibodies?

When encountering issues with Phospho-GATA4 (Ser105) antibody detection, consider these troubleshooting approaches:

For optimal results, recommended antibody dilutions are: WB (1:500-1:2000), IF/ICC (1:100-1:500), IHC (1:50-1:200), and ELISA (1:20000) .

How can Phospho-GATA4 (Ser105) antibodies be used to study cardiac development and pathology?

Phospho-GATA4 (Ser105) antibodies enable sophisticated investigations into cardiac biology:

• Developmental timing: Track GATA4 phosphorylation status during critical windows of cardiac development to correlate with gene expression changes. This approach has revealed that Ser105 phosphorylation peaks during chamber formation and trabeculation stages .

• Cellular stress responses: Monitor dynamic changes in GATA4 phosphorylation following hypoxia, oxidative stress, or mechanical stretch in cardiomyocytes. These studies have shown that rapid increases in Ser105 phosphorylation often precede the activation of cardioprotective gene programs .

• Disease modeling: Compare phosphorylation patterns between normal and pathological cardiac tissues (hypertrophy, ischemia, heart failure) to identify dysregulation of GATA4 activity. Recent research has demonstrated hyperphosphorylation of GATA4 at Ser105 in samples from patients with hypertrophic cardiomyopathy .

• Regenerative medicine: Assess GATA4 phosphorylation during differentiation of stem cells into cardiomyocytes, providing a molecular marker for cardiac lineage commitment. This application has become essential in protocols for generating functional cardiomyocytes from pluripotent stem cells .

• Drug discovery: Screen compounds that modulate GATA4 phosphorylation as potential therapeutics for heart disease, using high-throughput ELISA-based approaches with the phospho-specific antibody .

How does Phospho-GATA4 (Ser105) detection compare between different experimental models of cardiac hypertrophy?

Phospho-GATA4 (Ser105) detection reveals distinct patterns across cardiac hypertrophy models:

These differences highlight how the same phosphorylation event can participate in both adaptive and maladaptive cardiac remodeling depending on the context, duration, and accompanying signals. Careful experimental design is essential when comparing across these models, with particular attention to the timing of sample collection relative to the stimulus .

What methodological approaches can integrate Phospho-GATA4 (Ser105) antibody detection with other molecular techniques?

Advanced research integrates Phospho-GATA4 (Ser105) antibody detection with complementary techniques:

• ChIP-seq combined with phospho-GATA4 immunoprecipitation: This approach identifies genomic regions bound by specifically phosphorylated GATA4, revealing how phosphorylation alters the transcriptional profile. Recent studies have shown that Ser105 phosphorylation redirects GATA4 to different chromatin regions than unphosphorylated GATA4 .

• Proximity ligation assay (PLA): This technique detects in situ interactions between phosphorylated GATA4 and other cardiac transcription factors (like NKX2-5 or TBX5), visualizing specific protein complexes that form only when GATA4 is phosphorylated at Ser105 .

• FRET-based biosensors: Creating FRET sensors incorporating phospho-specific antibody fragments allows real-time monitoring of GATA4 phosphorylation dynamics in living cells, revealing oscillatory patterns following stimulation .

• Phosphoproteomics integration: Combining phospho-specific antibody detection with mass spectrometry-based phosphoproteomics provides comprehensive understanding of the signaling networks upstream and downstream of GATA4 phosphorylation .

• Single-cell analysis: Using flow cytometry or imaging cytometry with phospho-GATA4 antibodies can identify heterogeneity in cardiomyocyte populations, revealing subset-specific responses to cardiac stress .

For multiparametric analysis, careful optimization of fixation and permeabilization protocols is essential to maintain both phospho-epitope accessibility and cellular architecture .

How is Phospho-GATA4 (Ser105) analysis contributing to understanding cardiac regeneration?

Recent research using Phospho-GATA4 (Ser105) antibodies has provided crucial insights into cardiac regeneration:

• Regenerative capacity correlation: Studies in neonatal mice have revealed that high levels of phosphorylated GATA4 at Ser105 correlate with the limited window of mammalian cardiac regenerative capacity. Phospho-GATA4 levels decrease sharply after postnatal day 7, coinciding with the loss of regenerative potential .

• Cell cycle re-entry: Experimental manipulation of ERK signaling to maintain GATA4 Ser105 phosphorylation has been shown to enhance cardiomyocyte proliferation in adult tissues. The phosphorylated form specifically activates a subset of cell cycle genes through interaction with E2F transcription factors .

• Fibroblast reprogramming: During direct reprogramming of fibroblasts to cardiomyocytes, dynamic changes in GATA4 phosphorylation mark successful lineage conversion. Phospho-GATA4 (Ser105) appears early in the reprogramming process and precedes the expression of cardiac structural genes .

• Zebrafish model insights: Comparative studies using the antibody in zebrafish hearts (which regenerate throughout life) show persistently higher levels of phosphorylated GATA4 after injury compared to mammalian hearts, suggesting evolutionary divergence in this regulatory mechanism .

• Therapeutic targeting: Small molecules that indirectly promote GATA4 Ser105 phosphorylation by modulating upstream kinases have shown promise in enhancing recovery after myocardial infarction in preclinical models .

What are the methodological considerations when using Phospho-GATA4 (Ser105) antibodies in single-cell analyses?

Single-cell analysis with Phospho-GATA4 (Ser105) antibodies requires specialized approaches:

• Fixation optimization: For flow cytometry or mass cytometry (CyTOF), a dual fixation protocol yields better results:

  • Brief formaldehyde fixation (10 minutes, 4% PFA)

  • Followed by methanol permeabilization (-20°C, 30 minutes)

  • This preserves both phospho-epitope integrity and cellular architecture

• Signal amplification: For detecting low-abundance phospho-signals in individual cells:

  • Tyramide signal amplification for immunofluorescence

  • Metal-conjugated secondary antibodies for mass cytometry

  • Both approaches can improve detection sensitivity up to 100-fold

• Multiplexing considerations: When combining with other antibodies:

  • Sequential staining may be necessary to avoid epitope masking

  • Careful antibody panel design to minimize spectral overlap

  • Inclusion of phosphorylation-state negative controls in each experiment

• Timing of analysis: Phosphorylation states are dynamic and can change rapidly:

  • Standardize time from stimulus to fixation

  • Consider using phosphatase inhibitors in all buffers

  • Include time-course experiments to capture transient states

• Data normalization: For quantitative comparisons:

  • Normalize phospho-signal to total GATA4 levels when possible

  • Include internal phosphorylation standards

  • Apply appropriate statistical methods for bimodal distributions often seen with phospho-proteins

These methodological refinements enable researchers to detect cell-to-cell variability in GATA4 phosphorylation status, revealing previously unappreciated heterogeneity in cardiac cellular responses to stress and developmental signals .

How should researchers interpret contradictory results between different phospho-specific GATA4 antibodies?

When faced with contradictory results using different phospho-specific GATA4 antibodies, researchers should implement a systematic validation approach:

• Epitope mapping comparison: Different antibodies may recognize slightly different epitopes surrounding Ser105. Some may be affected by neighboring phosphorylation events or conformational changes. Compare the immunogen sequences between antibodies to identify potential differences .

• Cross-reactivity profiling: Test each antibody against recombinant GATA4 proteins with single or multiple phosphorylation sites to determine specificity profiles. Some antibodies may detect phosphorylation at similar motifs in other GATA family members .

• Validation across techniques: An antibody that works well in Western blot may not perform equivalently in IHC or IP applications due to differences in protein conformation and epitope accessibility. Validate each antibody in the specific application context .

• Knockout/knockdown controls: Include GATA4 knockout or knockdown samples as definitive negative controls. Any signal in these samples indicates non-specific binding .

• Phosphatase treatment gradients: Treat samples with increasing concentrations of phosphatase to create a gradient of dephosphorylation. Truly phospho-specific antibodies will show proportional signal reduction .

• Correlation with kinase activity: Validate results by correlating antibody signals with direct measurements of upstream kinase activity (ERK1/2 for Ser105). Concordance increases confidence in antibody specificity .

What are the critical controls needed when studying GATA4 phosphorylation in disease models?

Robust experimental design for studying GATA4 phosphorylation in disease models requires comprehensive controls:

The integration of these controls allows researchers to distinguish genuine pathological changes in GATA4 phosphorylation from technical artifacts or secondary consequences of disease .

How might single-molecule techniques enhance our understanding of GATA4 phosphorylation dynamics?

Emerging single-molecule techniques offer unprecedented insights into GATA4 phosphorylation dynamics:

• Single-molecule tracking: Using fluorescently-tagged phospho-specific antibody fragments (Fabs) to track individual phosphorylated GATA4 molecules in living cells can reveal:

  • Real-time phosphorylation events

  • Nuclear diffusion rates before and after phosphorylation

  • Residence times at target chromatin sites

  • These measurements could demonstrate how phosphorylation alters GATA4's search mechanism for DNA targets

• Super-resolution microscopy: Techniques like STORM or PALM with phospho-specific antibodies can visualize:

  • Nanoscale clustering of phosphorylated GATA4 molecules

  • Co-clustering with specific transcriptional partners

  • Localization relative to nuclear structures like transcription factories

  • Recent applications have shown that phosphorylated GATA4 forms distinct nuclear subdomains during hypertrophic responses

• FRET-FLIM sensors: Genetically-encoded FRET sensors for GATA4 phosphorylation can detect:

  • Spatiotemporal dynamics of phosphorylation within different nuclear regions

  • Oscillatory patterns of phosphorylation/dephosphorylation

  • Transmission of phosphorylation waves across cells in cardiac tissue

  • These approaches have recently revealed unexpected compartmentalization of GATA4 phosphorylation events

• Single-molecule pull-down (SiMPull): This technique can quantify:

  • Exact stoichiometry of phosphorylated GATA4 in transcriptional complexes

  • Order of assembly of multi-protein complexes involving phospho-GATA4

  • Binding affinities altered by phosphorylation status

  • Preliminary studies suggest phosphorylation creates specific interaction nodes in cardiac transcriptional networks

These advanced techniques promise to transform our understanding from static snapshots to dynamic models of GATA4 regulation in cardiac biology .

What are potential translational applications of Phospho-GATA4 (Ser105) detection in cardiac medicine?

Phospho-GATA4 (Ser105) detection holds promising translational applications:

• Biomarker development: Phospho-GATA4 levels in circulating nucleic acid-binding proteins or exosomes could serve as biomarkers for:

  • Early cardiac stress responses before troponin elevation

  • Monitoring efficacy of heart failure treatments

  • Predicting cardiac remodeling outcomes after myocardial infarction

  • Preliminary studies have identified circulating phosphorylated transcription factors in patient blood samples following cardiac events

• Therapeutic target identification: Screening compounds that modulate GATA4 phosphorylation could:

  • Identify novel cardioprotective agents

  • Discover drugs that promote cardiomyocyte proliferation

  • Develop treatments for specific congenital heart defects linked to GATA4 mutations

  • Recent high-throughput screens have identified small molecules that selectively modulate GATA4 activity states

• Personalized medicine approaches: Analyzing patient-derived cardiac samples for phospho-GATA4 patterns might:

  • Stratify heart failure patients into treatment-responsive subgroups

  • Guide timing of interventions based on molecular rather than clinical criteria

  • Tailor therapeutic strategies to individual phosphorylation profiles

  • Early clinical studies have shown heterogeneity in GATA4 phosphorylation patterns among seemingly similar heart failure patients

• Regenerative medicine applications: Manipulating GATA4 phosphorylation could optimize:

  • Differentiation protocols for cardiac tissue engineering

  • Cell therapy approaches using enhanced cardiomyocyte progenitors

  • Direct reprogramming efficiency for cardiac regeneration

  • Preliminary work shows that timed modulation of ERK signaling improves cardiac differentiation outcomes

These translational directions represent the frontier where basic research on GATA4 phosphorylation meets clinical cardiology needs .