Phospho-GATA4 (Ser262) Antibody

What is the biological significance of GATA4 Ser262 phosphorylation?

GATA4 is a critical transcription factor that plays pivotal roles in cardiac development, differentiation, and proliferation. Phosphorylation of GATA4 at serine 262 represents a key regulatory mechanism that modulates its transcriptional activity .

Research has demonstrated that this specific phosphorylation event:

• Is mediated primarily by Protein Kinase A (PKA) in response to cAMP signaling in gonadal cells

• Enhances GATA4's transcriptional activity on different gonadal promoters

• Leads to enhanced recruitment of CREB-binding protein coactivator

• Occurs at an evolutionarily conserved serine residue (position 261 in some species)

This phosphorylation event is particularly significant as dysregulation of GATA4 phosphorylation can contribute to various cardiac diseases, making it a valuable target for therapeutic interventions .

What signaling pathways are known to regulate GATA4 Ser262 phosphorylation?

GATA4 Ser262 phosphorylation is primarily regulated through the cAMP/PKA signaling pathway. Studies have shown that:

• cAMP stimulation of gonadal cells leads to rapid phosphorylation of GATA4

• This phosphorylation can be blocked by PKA-specific inhibitors but not by mitogen-activated protein kinase (MAPK) inhibitors

• PKA phosphorylates GATA4 predominantly at the evolutionarily conserved serine residue (position 261/262)

• While ERK2, p38α, and JNK1 can potentially phosphorylate GATA4 in vitro , the primary kinase responsible for Ser262 phosphorylation in vivo appears to be PKA

This pathway is particularly important in contexts like gonadal gene expression regulation by pituitary hormones, where GATA4 serves as a downstream effector of cAMP/PKA signaling .

How does phosphorylation at Ser262 affect GATA4 function?

Phosphorylation of GATA4 at Ser262 significantly modifies its functional capacity in several ways:

• Enhanced transcriptional activity: Phosphorylation at Ser262 increases GATA4's transcriptional activity on various gonadal promoters

• Coactivator recruitment: This phosphorylation leads to enhanced recruitment of the CREB-binding protein (CBP) coactivator

• Synergistic interactions: Phospho-GATA4 shows altered synergistic activity with other transcription factors, including NKX2-5, which is crucial for cardiac gene expression

• Cardiac development: Altered phosphorylation status at this site can impact cardiac development and function

Functional studies using S261A mutants (preventing phosphorylation) have demonstrated markedly reduced transcriptional activity and blunted cAMP-induced promoter activity in gonadal cells, confirming the importance of this phosphorylation site .

What are the key applications of Phospho-GATA4 (Ser262) antibodies in research?

Phospho-GATA4 (Ser262) antibodies serve multiple research applications:

• Western Blot analysis: For detecting and quantifying phosphorylated GATA4 levels in cell or tissue lysates

• Immunofluorescence/Immunocytochemistry: For visualizing cellular localization of phosphorylated GATA4

• ELISA assays: For quantitative analysis of phosphorylated GATA4 levels

• Cell-Based Colorimetric Assays: For measuring relative amounts of phosphorylated GATA4 in cultured cells

• Transcription factor activity assays: For detecting and quantifying GATA4 phosphorylation and its impact on transcriptional activity

These antibodies are particularly valuable for:

• Studying cardiac development and disease mechanisms

• Investigating gonadal gene expression regulation

• Examining the effects of various treatments or inhibitors on GATA4 phosphorylation

• Understanding the molecular mechanisms of cAMP/PKA signaling pathways

What controls should be included when using Phospho-GATA4 (Ser262) antibodies?

When working with Phospho-GATA4 (Ser262) antibodies, the following controls are essential for robust experimental design:

Positive controls:

• Lysates from cells treated with PKA activators (e.g., forskolin or cAMP analogs)

• Recombinant phosphorylated GATA4 protein (if available)

• Cells transfected with wild-type GATA4 and treated with PKA activators

Negative controls:

• Antigen-specific peptide blocking: The antibody should be pre-incubated with the phosphopeptide immunogen to confirm specificity

• Phosphatase treatment: Sample treatment with lambda phosphatase to remove phosphorylation

• GATA4 S261A/S262A mutant-expressing cells that cannot be phosphorylated at this site

• Samples treated with PKA inhibitors to prevent phosphorylation

Additional controls:

• Total GATA4 antibody detection in parallel samples to normalize phospho-signal to total protein

• Use of unrelated phospho-specific antibodies as a specificity control

• Testing in GATA4 knockout/knockdown systems

As seen in validation studies, phospho-specific antibodies should detect reduced or absent signal when treated with the antigen-specific peptide, confirming their specificity .

What are the optimal sample preparation methods for detecting phosphorylated GATA4?

Optimal sample preparation is critical for preserving phosphorylation status and achieving reliable results:

Cell/Tissue Lysis:

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

• Include protease inhibitors to prevent protein degradation

• Maintain samples at 4°C throughout processing

• For nuclear proteins like GATA4, consider using specific nuclear extraction protocols

Buffer Composition:

• Recommended buffer: PBS containing phosphatase inhibitors, protease inhibitors, 0.5% BSA (for stabilization)

• For storage, include 50% glycerol and 0.02% sodium azide

Sample Handling:

• Process samples quickly to minimize phosphorylation loss

• Avoid repeated freeze-thaw cycles

• Store samples at -80°C for long-term preservation

• For Western blotting, use freshly prepared samples when possible

Protein Quantification:

• Use Bradford or BCA assays to ensure equal loading

• Load 20-40 μg of total protein per lane for Western blotting

• For cardiac tissue samples, special attention to extraction methods may be needed due to high background from structural proteins

These methods have been validated in studies examining GATA4 phosphorylation in various cell types and tissues .

How can Phospho-GATA4 (Ser262) antibodies be validated for specificity in experimental settings?

Rigorous validation of phospho-specific antibodies is crucial for reliable research. For Phospho-GATA4 (Ser262) antibodies, consider these validation strategies:

Peptide Competition Assays:

• Pre-incubate the antibody with the phosphorylated peptide immunogen

• A specific antibody will show significantly reduced or eliminated signal

• Include non-phosphorylated peptide as a control that should not block specific binding

Genetic Approaches:

• Use GATA4 S261A/S262A mutants where the serine is replaced with alanine

• These mutants cannot be phosphorylated at the target site and should show no signal

• Complementary approach: overexpress wild-type GATA4 and observe increased signal

Pharmacological Approaches:

• Treat samples with PKA activators (forskolin, cAMP analogs) to increase phosphorylation

• Use PKA inhibitors to block phosphorylation as negative controls

• Treat samples with lambda phosphatase to remove phosphorylation

Verification Across Techniques:

• Confirm findings using multiple techniques (Western blot, immunofluorescence, ELISA)

• Use alternative antibodies targeting the same phosphorylation site from different vendors

• Compare results with functional assays that measure GATA4 transcriptional activity

Example data from validation studies show that in Western blot analysis of 293 cell extracts, specific bands are detected with the Phospho-GATA4 (Ser262) antibody that disappear when the antibody is pre-treated with the antigen-specific peptide .

How does GATA4 Ser262 phosphorylation interact with other post-translational modifications?

GATA4 undergoes multiple post-translational modifications that can interact with Ser262 phosphorylation in complex ways:

Interactions with other phosphorylation sites:

• GATA4 can be phosphorylated by multiple kinases including PKA, MAPK, and GSK3β

• ERK2 phosphorylation of GATA4 may work in concert with or independently of PKA-mediated Ser262 phosphorylation

• Sequential phosphorylation patterns may exist where one modification influences the likelihood of others

Functional cross-talk:

• Phosphorylation at Ser262 enhances CBP coactivator recruitment

• This may interact with acetylation states of GATA4, as CBP has acetyltransferase activity

• The synergistic activity between GATA4 and NKX2-5 is modulated by phosphorylation status

Regulatory hierarchy:

• PKA-mediated phosphorylation at Ser262 appears to be a primary regulatory event in gonadal cells

• This may set the stage for subsequent modifications in a temporal sequence

• Different cell types may show distinct patterns of modification cross-talk

What are the challenges in quantifying GATA4 Ser262 phosphorylation levels in primary cardiac tissues?

Quantifying GATA4 Ser262 phosphorylation in primary cardiac tissues presents several methodological challenges:

Tissue heterogeneity:

• Cardiac tissue contains multiple cell types (cardiomyocytes, fibroblasts, endothelial cells)

• GATA4 expression and phosphorylation patterns may vary between these cell types

• Cell-type specific isolation or single-cell approaches may be needed for precise quantification

Rapid phosphorylation dynamics:

• Phosphorylation states can change rapidly during tissue collection and processing

• Immediate preservation of phosphorylation status is critical (flash freezing, phosphatase inhibitors)

• Time from tissue collection to fixation/extraction must be minimized and standardized

Background interference:

• Cardiac tissues have high levels of structural proteins that can interfere with detection

• Autofluorescence can complicate immunofluorescence studies

• Specialized extraction protocols may be required to achieve clean nuclear fraction isolation

Quantification considerations:

• Normalization to total GATA4 is essential but may be complicated by antibody affinity differences

• Phosphorylation levels may change with cardiac disease states or development

• Standard curves using recombinant phosphorylated proteins are recommended for absolute quantification

Recommended approach:

• Use a combination of techniques (Western blot, ELISA, immunohistochemistry)

• Include appropriate controls for each experiment

• Consider phospho-enrichment methods prior to analysis

• Validate findings across multiple experimental models and techniques

How can Phospho-GATA4 (Ser262) antibodies be used in ChIP experiments?

Chromatin Immunoprecipitation (ChIP) with Phospho-GATA4 (Ser262) antibodies allows researchers to study how phosphorylation affects DNA binding and target gene regulation:

Protocol considerations:

• Crosslinking: Use 1% formaldehyde for 10 minutes at room temperature

• Sonication: Optimize conditions to achieve chromatin fragments of 200-500bp

• Immunoprecipitation: Use 3-5μg of Phospho-GATA4 (Ser262) antibody per reaction

• Controls: Include IgG control, input control, and when possible, a non-phosphorylatable GATA4 mutant

• Washing: Use stringent washing conditions to reduce background

Target selection:

• Known GATA4 binding sites in cardiac genes (ANF, BNP)

• GATA consensus sequences (5'-AGATAG-3')

• Regions co-regulated by GATA4 and NKX2-5

Data analysis approach:

• Compare binding profiles of phosphorylated vs. total GATA4

• Analyze how stimuli that increase phosphorylation affect binding patterns

• Integrate with RNA-seq data to correlate binding with transcriptional outcomes

Special considerations:

• Pre-clear chromatin with protein A/G beads to reduce background

• Use fresh antibody preparations for optimal results

• Consider dual ChIP to study co-localization with interacting factors like NKX2-5 or CREB-binding protein

This approach allows researchers to determine whether phosphorylation at Ser262 alters GATA4's genomic binding profile and target gene selection.

What are the differences between various commercial Phospho-GATA4 (Ser262) antibodies?

Commercial Phospho-GATA4 (Ser262) antibodies vary in several key characteristics that can impact experimental outcomes:

Antibody formats and characteristics:

Performance considerations:

• Reactivity: Most antibodies react with human, mouse, and rat GATA4

• Dilution recommendations vary by application:

  • Western Blot: 1:500-1:2000

  • Immunofluorescence: 1:100-1:1000

  • ELISA: 1:5000

• Storage conditions: Most require -20°C storage in buffer containing 50% glycerol

Validation data:

• Western blot validation typically shows a band at approximately 48 kDa

• Most manufacturers provide data showing specificity using peptide competition assays

• Some provide phosphatase treatment validation

When selecting an antibody, researchers should consider their specific application needs, species of interest, and available validation data most relevant to their experimental system.

How does the role of GATA4 Ser262 phosphorylation differ between cardiac and gonadal tissues?

GATA4 Ser262 phosphorylation exhibits tissue-specific functions and regulatory mechanisms:

Cardiac tissues:

• Phosphorylation of GATA4 at Ser262 modulates cardiac development and function

• This modification affects the synergistic interaction between GATA4 and NKX2-5, critical for cardiac gene expression

• In cardiac cells, GATA4 phosphorylation promotes cardiomyocyte gene expression while downregulating endocardial and endothelial gene expression

• GATA4 phosphorylation is linked to cardiac hypertrophy responses and may contribute to pathological states

Gonadal tissues:

• In gonadal cells, GATA4 Ser262 phosphorylation is primarily mediated by the cAMP/PKA pathway

• This phosphorylation enhances transcriptional activity on gonadal promoters

• It serves as a mechanism for conveying cAMP responsiveness to gonadal genes that lack classical cAMP regulatory elements

• The modification is critical for hormone-regulated gene expression in reproductive tissues

Comparative analysis:

• In both tissues, phosphorylation enhances GATA4's transcriptional activity

• The upstream signaling differs: predominantly PKA in gonadal tissues vs. potentially multiple kinases in cardiac tissues

• Downstream gene targets are tissue-specific despite the same phosphorylation site

• Interacting partners may differ, though CBP recruitment is enhanced in both contexts

This tissue-specific role highlights the importance of cellular context in determining the functional outcome of the same post-translational modification on GATA4.

What are common troubleshooting issues when using Phospho-GATA4 (Ser262) antibodies?

Researchers commonly encounter several challenges when working with Phospho-GATA4 (Ser262) antibodies:

High background in Western blots:

• Cause: Insufficient blocking, antibody concentration too high, or non-specific binding

• Solution: Increase blocking time (5% BSA in TBST recommended), optimize antibody dilution (start with 1:1000) , use more stringent washing steps

Weak or no signal:

• Cause: Rapid dephosphorylation during sample handling, insufficient protein, or antibody degradation

• Solution: Ensure robust phosphatase inhibition throughout all steps, increase protein loading (40-50μg recommended), use fresh antibody dilutions

Multiple bands or unexpected molecular weight:

• Cause: Cross-reactivity, protein degradation, or post-translational modifications

• Solution: Validate with peptide competition , include protease inhibitors, compare with total GATA4 antibody pattern

Poor immunostaining results:

• Cause: Inadequate fixation, epitope masking, or phosphatase activity during processing

• Solution: Optimize fixation protocol (4% paraformaldehyde recommended), include phosphatase inhibitors in all buffers, try antigen retrieval methods

Inconsistent results between experiments:

• Cause: Variation in phosphorylation levels due to cell culture conditions or handling

• Solution: Standardize cell culture conditions, ensure consistent stimulation/inhibition protocols, process all comparative samples simultaneously

Quantification challenges:

• Cause: Variable total GATA4 expression between samples

• Solution: Always normalize phospho-signal to total GATA4 levels detected on parallel blots or after membrane stripping and reprobing

Implementing these troubleshooting strategies will improve the reliability and reproducibility of experiments using Phospho-GATA4 (Ser262) antibodies.

How do different fixation methods affect Phospho-GATA4 (Ser262) antibody performance in immunohistochemistry?

Fixation methods significantly impact phospho-epitope preservation and antibody accessibility in immunohistochemical applications:

Comparison of fixation methods:

Critical factors for phospho-epitope preservation:

• Timing: Minimize time between tissue collection and fixation

• Temperature: Keep samples cold prior to fixation

• Inhibitors: Include phosphatase inhibitors in all solutions

• pH: Maintain neutral pH during fixation (pH 7.2-7.4)

Antigen retrieval for FFPE tissues:

• Heat-induced epitope retrieval in citrate buffer (pH 6.0, 95°C for 20 min) shows best results

• Enzymatic retrieval methods typically show poor results with phospho-epitopes

• For Phospho-GATA4 (Ser262), EDTA buffer (pH 8.0) may provide superior results in some tissues

Recommendations:

• For cultured cells, 4% paraformaldehyde for 15 minutes at room temperature gives optimal results

• For frozen tissue sections, brief fixation (10 min) in cold 4% paraformaldehyde is recommended

• For FFPE sections, extended heat-induced epitope retrieval is essential

• Always run parallel sections with phosphatase-treated controls to confirm specificity

These considerations will help researchers optimize immunohistochemical detection of Phospho-GATA4 (Ser262) across different sample types and fixation methods.

How can researchers effectively study the functional consequences of GATA4 Ser262 phosphorylation?

To comprehensively investigate the functional impact of GATA4 Ser262 phosphorylation, researchers should employ multiple complementary approaches:

Genetic modulation approaches:

• Generate phospho-deficient mutants (S262A) to prevent phosphorylation

• Create phospho-mimetic mutants (S262D/S262E) to simulate constitutive phosphorylation

• Use CRISPR/Cas9 to introduce these mutations in cellular or animal models

• Develop conditional knock-in models to study tissue-specific effects

Pharmacological manipulation:

• Modulate PKA activity using activators (forskolin, cAMP analogs) and inhibitors

• Target upstream regulators of the cAMP/PKA pathway

• Use temporal control of phosphorylation through acute drug treatments

• Combine with phospho-specific antibodies to confirm target engagement

Transcriptional output assessment:

• Luciferase reporter assays with GATA4 target promoters

• ChIP-seq to map genome-wide binding profiles of phosphorylated vs. non-phosphorylated GATA4

• RNA-seq to identify differentially expressed genes

• Measure synergistic activity with co-factors like NKX2-5

Protein-protein interaction studies:

• Co-immunoprecipitation using phospho-specific antibodies

• Proximity ligation assays to detect interactions in situ

• Assess CBP/p300 recruitment, which is enhanced by S262 phosphorylation

• Mass spectrometry to identify phosphorylation-dependent interactome changes

Functional readouts in relevant systems:

• In cardiac cells: hypertrophic response, contractility, calcium handling

• In gonadal cells: hormone responsiveness, steroidogenesis

• In disease models: impact on pathological progression

• In developmental models: effects on organogenesis and differentiation

An integrated approach combining these methods will provide comprehensive insights into how this specific phosphorylation event controls GATA4 function in different biological contexts.