Phospho-GATA2 (S401) Antibody
Description
Introduction to Phospho-GATA2 (S401) Antibody
The Phospho-GATA2 (S401) Antibody is a monoclonal or polyclonal antibody that selectively binds to the phosphorylated Ser401 residue of the GATA2 protein. This modification regulates GATA2’s transcriptional activity, subcellular localization, and interaction with DNA . GATA2 is a zinc-finger transcription factor critical for hematopoiesis, endothelial cell function, and immune regulation . Phosphorylation at Ser401, mediated by kinases such as Akt, modulates its role in adipogenesis, inflammation, and macrophage-like functions in preadipocytes .
Key Technical Attributes
Key Features
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Specificity: Recognizes endogenous GATA2 only when phosphorylated at Ser401, validated via kinase assays and mutagenesis .
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Applications: Primarily used in Western blotting (1:500–1:2000 dilution) and ELISA (1:5000 dilution) .
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Validation: Confirmed through immunofluorescence, showing reduced nuclear GATA2 upon phosphorylation .
Mechanistic Insights
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Akt-Mediated Phosphorylation:
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Functional Consequences:
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Subcellular Localization: Phosphorylated GATA2 (S401D mutant) accumulates in the cytoplasm, while the non-phosphorylatable mutant (S401A) remains nuclear .
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Phagocytic Activity: Preadipocytes expressing S401A GATA2 exhibit increased phagocytosis and cytokine production (e.g., MCP-1, GM-CSF) compared to S401D mutants .
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Inflammatory Signaling: Unphosphorylated GATA2 promotes macrophage-like behavior in preadipocytes, linking it to adipose tissue inflammation .
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Key Experimental Data
| Experiment | S401A Mutant (Non-Phospho) | S401D Mutant (Phospho-Mimetic) |
|---|---|---|
| Nuclear Localization | Retained in nucleus | Cytoplasmic accumulation |
| DNA Binding | High affinity | Reduced binding |
| Phagocytosis | Increased activity | Suppressed activity |
| Cytokine Expression | ↑ MCP-1, GM-CSF, TPO, IL-4 | ↓ Inflammatory markers |
Implications in Disease and Therapeutic Contexts
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Adipose Tissue Inflammation: Phospho-GATA2 (S401) is implicated in monocyte recruitment to adipose tissue, contributing to metabolic disorders .
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Hematopoietic Disorders: GATA2 mutations drive myeloid malignancies (e.g., MDS/AML) and immunodeficiency . While phosphorylation at Ser401 is not directly linked to these conditions, its regulatory role in transcriptional activity suggests potential therapeutic modulation .
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Pulmonary Disease: GATA2 deficiency causes alveolar proteinosis and infections; phosphorylation status may influence macrophage dysfunction .
Product Specs
Target Background
- Two GATA2 mutants (gT354M and gC373R) bound the key hematopoietic differentiation factor PU.1. PMID: 28642594
- Research has identified the GATA2 transcription factor as a crucial regulator of matrix stiffness induced transcriptional program in lymphatic endothelial cells (LECs). Contrary to previous findings that suggested activation of GATA2 by increased mechanical stimulus upon exposure of LECs to stiff matrix or oscillatory flow, this study found that GATA2 expression is elevated in LECs grown on a soft matrix. PMID: 29666442
- Endothelial SENP1-mediated SUMOylation drives graft arteriosclerosis by regulating the synergistic effect of GATA2 and NF-kappaB and consequent endothelial dysfunction. PMID: 28569748
- The aim of this study was to analyze the outcomes of COS in women with GATA2 deficiency. PMID: 29532200
- Data suggest that the stress of intermittent hypoxia up-regulates expression of mRNA and protein for POMC and CART in neuronal cell lines; GATA2 and GATA3 appear to be involved in these stress mechanisms. (POMC = proopiomelanocortin; CART = cocaine- and amphetamine-regulated transcript protein; GATA2 = endothelial transcription factor GATA-2; GATA3 = T-cell-specific transcription factor GATA-3) PMID: 29275211
- Gata2 and miR-124-3p are potential novel reporter biomolecules for ovarian cancer. PMID: 28937943
- While relatively infrequent in patients with hypertrophic cardiomyopathy, GATA2, 4 and 6 transcription factors may represent a novel insight into the molecular mechanisms related to the pathogenesis of hypertrophic cardiomyopathy. PMID: 28381408
- In our study on a large cohort of CEBPAmut AML patients, we observed a high coincidence of GATA2mut, particularly within the subgroup of patients with CEBPAbi mutations. PMID: 27375010
- Androgen receptor transcriptionally regulates semaphorin 3C in a GATA2-dependent manner in prostate tumor cells. PMID: 28038451
- This study identified the novel role of transcription factors GATA-2 and GATA-3 in suppressing MICA/B expression in HBV-infected human hepatoma cells. PMID: 27528231
- GATA2 mutation-related immunodeficiency may predispose to Epstein-Barr virus-associated subacute demyelinating polyradiculoneuropathy by both viral susceptibility and immune dysregulation. PMID: 28093780
- The absence of deleterious mutations in this large cohort of familial aggregations of hematological malignancies may strengthen the hypothesis that GATA2 mutations are a significant predisposing factor, though potentially as a secondary genetic event, required for the development of overt malignant disease. PMID: 28752392
- Gata2 regulates a key regulatory network of gene expression for progesterone signaling at the early pregnancy stage. PMID: 27783953
- A p38-dependent mechanism that phosphorylates GATA-2 and increases GATA-2 target gene activation has been demonstrated. This mechanism establishes a growth-promoting chemokine/cytokine circuit in acute myeloid leukemia cells. PMID: 27545880
- We report a family with a null mutation in GATA2 and Emberger syndrome with variable expressivity and incomplete penetrance. This case highlights the need for considering genetic interactions to produce specific phenotypes in the clinical spectrum of GATA2 deficiency, where a precise genotype-phenotype correlation has not been established. PMID: 28271814
- This study investigated GATA2 Deficiency With Severe Primary Epstein-Barr Virus (EBV) Infection and EBV-associated Cancers. Seven patients with GATA2 deficiency developed severe EBV disease. The study also identified GATA2 as the first gene associated with EBV hydroa vacciniforme-like lymphoma. PMID: 27169477
- GATA2 deficiency is associated with impaired membrane expression and chemotactic dysfunctions of CXCR4. PMID: 26710799
- The decline of GATA2 resulting from mutations contributes to the erythroid commitment, differentiation and the development of AEL. PMID: 27389056
- This study investigated the distinct mechanisms by which GATA2 and FOXA1 regulate AR cistrome and suggests that FOXA1 acts upstream of GATA2 and AR in determining hormone-dependent gene expression in prostate cancer. PMID: 26751772
- Adaptive NK cells can persist in patients with GATA2 mutation, even after NK-cell progenitors expire. PMID: 28209719
- Reducing GATA2 expression or inhibition of its transcription activity can alleviate the drug resistance of acute myeloid leukemia cells, potentially aiding in the elimination of leukemia cells in patients. PMID: 28114350
- We recommend screening for GATA2 mutations in pediatric myelodysplastic syndrome, particularly in patients with impaired B-cell homeostasis in bone marrow and peripheral blood (low number of progenitors, intronRSS-Kde recombination excision circles and naive cells). PMID: 27013649
- Knockdown of GATA-2 caused NGB expression to drop dramatically, indicating GATA-2 as an essential transcription factor for the activation of NGB expression via binding to a novel distal regulatory element of NGB. PMID: 27651453
- This study analyzed two separate cases of pediatric AML/MDS with underlying GATA2 mutations who underwent a successful umbilical cord hematopoietic stem cell transplantation using two different conditioning regimens [case report/review]. PMID: 27416790
- Germ line mutations in GATA2 are associated with GATA2 deficiency syndrome, while acquired mutations are seen in myelodysplastic syndrome, acute myeloid leukemia, and in blast crisis transformation of chronic myeloid leukemia. PMID: 28179280
- The results of this study demonstrated that high expression of GATA2 is correlated with worse survival outcomes in KRAS mutant colorectal cancer patients. PMID: 27460045
- Letter/Case Report: frameshift mutation in GATA2 resulting in Emberger syndrome in Korean patient. PMID: 26767875
- GATA2 was differentially expressed between Fibromyalgia patients and healthy controls. PMID: 27157394
- We screened 995 transcription factor genes and revealed that CITED2 acts as a GATA-2 activator in human hematopoietic cells. These findings provide novel insights into and further identify the regulatory mechanism of GATA-2. PMID: 26325290
- Results demonstrated that GATA2 single nucleotide polymorphism rs2335052 is an independent predictor for prognosis of colorectal cancer patients. PMID: 26287967
- Data show that the Lim domain only 2 (LMO2) regulatory element (element-25) region consists of transcription factor GATA2-binding myeloid ennhancer and RUNX protein-binding T-cell repressor. PMID: 26161748
- High GATA2 promotes glioma progression through EGFR/ERK/Elk-1 pathway. PMID: 25707769
- In children and adults with severe GM-CSF negative PAP, close collaboration between pneumologists and hemato-oncologists is needed to diagnose the underlying diseases, some of which are caused by mutations of transcription factor GATA2. PMID: 26264606
- The results of this study indicated that GATA2 regulates the development of cochlear hair cells and the inferior colliculus (IC), which are important in tonotopic mapping. PMID: 24614497
- Germline mutation in GATA2 in a pedigree presenting with myelodysplastic syndrome/acute myeloid leukemia is associated with concurrent thrombocytopenia. PMID: 25676417
- Data unveil essential roles for GATA2 in the lymphatic vasculature and explain why a select catalogue of human GATA2 mutations results in lymphedema. PMID: 26214525
- Altered binding of GATA2 (and Sp1/TBP) mediates aberrant MAOA expression under pathophysiological conditions. PMID: 25810277
- Transgenic human GATA2 appeared to induce growth via downstream activation of Nmyc and Hoxa9 in mice. GATA2 overexpression at low level confers self-renewal capacity to myeloid progenitors and is relevant to myeloid leukemia development. PMID: 25907033
- This review discusses the molecular biology, clinical, hematological and immunological features of patients with GATA2 mutations. PMID: 25707267
- GATA2 plays a crucial role in regulating the differentiation potential of BM-MSC and contributes to hematopoietic supporting capacity. PMID: 25150255
- Homozygous CEPBA mutations have a similar gene expression signature as CEPBA double mutations and thus may be considered as equivalent in the presence of GATA2 mutations. PMID: 25611491
- Ubiquitin-dependent degradation of GATA 2 is promoted by Fbw7, is cyclin B-CDK1-mediated Thr176 phosphorylation-dependent, and influences hematopoietic cell differentiation. PMID: 25670854
- The study showed that human GATA2 binds to its own promoter, activating its transcription, and that the p.Arg396Gln mutation impairs the transcription of GATA2. PMID: 25624456
- One of the targets of p38 in this context is another GATA transcription factor, GATA3, recently demonstrated to promote HSC cycling and restrict their long-term reconstitutive potential downstream of p38 in stress-induced hematopoiesis [9]. PMID: 25092790
- p38 also induced multisite phosphorylation of wild-type GATA-2, which required a single phosphorylated residue (S192). Phosphorylation of GATA-2, but not T354M, stimulated target gene expression. PMID: 25056917
- These studies reveal a GATA2-IGF2 aggressiveness axis in lethal prostate cancer and identify a therapeutic opportunity in this challenging disease. PMID: 25670080
- Data suggest that the original GATA binding protein 2 (GATA2) mutations might be lost during disease progression in GATA2-mutated patients, while novel GATA2 mutations might be acquired at relapse in GATA2-wild patients. PMID: 25241285
- We propose pharmacological inhibition of GATA2 as a first-in-field approach to target AR expression and function and improve outcomes in CRPC. PMID: 25489091
- GATA2 deficiency analysis is crucial for the differential diagnosis of idiopathic aplastic anemia. PMID: 25359990
- GATA2 is epigenetically repressed in human and mouse lung tumors and its further inhibition is not a valid therapeutic strategy for KRAS mutant lung cancer. PMID: 24807155
Q&A
How do I validate the specificity of Phospho-GATA2 (S401) antibodies in experimental systems?
Methodological Answer
To validate specificity, use three complementary approaches:
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Peptide Blocking: Pre-incubate the antibody with its immunogen peptide (e.g., residues 367–416 of human GATA2 with phosphorylated Ser401 ). A ≥80% reduction in signal confirms specificity.
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Genetic Knockdown/Overexpression: Compare signals in wild-type vs. GATA2-knockout cell lines (e.g., CRISPR-edited HEK293) or cells overexpressing S401A/S401D mutants .
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Phosphatase Treatment: Treat lysates with λ-phosphatase; phospho-specific signals should disappear .
Table 1: Validation Parameters from Published Studies
What are optimal protocols for detecting phospho-GATA2 (S401) via Western blot?
Methodological Answer
Optimal conditions depend on cell type and activation state:
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Lysis Buffer: Use RIPA buffer with phosphatase inhibitors (e.g., 1 mM Na3VO4, 10 mM β-glycerophosphate) .
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Blocking: 5% BSA (not skim milk) to avoid nonspecific binding .
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Activation: Treat cells with TNFα (20 ng/mL, 5 min) or insulin (100 nM, 15 min) to induce phosphorylation .
Critical Note: Always include a non-phosphorylated GATA2 antibody (e.g., ab153820 ) as a loading control.
How to resolve contradictions in phospho-GATA2 (S401) localization data across studies?
Methodological Answer
Conflicting reports of nuclear vs. cytoplasmic localization arise from:
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Cellular Context: In adipocytes, insulin-induced S401 phosphorylation promotes cytoplasmic retention , while in hematopoietic cells, phosphorylation enhances nuclear activity .
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Fixation Artifacts: Use methanol-free formaldehyde (4%, 10 min) for immunofluorescence to preserve epitopes .
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Quantitative Imaging: Combine confocal microscopy with subcellular fractionation (e.g., NE-PER kit) and immunoblotting .
Table 2: Context-Dependent Localization Findings
| Cell Type | Treatment | Localization | Study |
|---|---|---|---|
| Preadipocytes | Insulin (100 nM) | Cytoplasmic | Circulation (2005) |
| Kasumi-1 AML cells | Oncogenic Ras | Nuclear | Blood (2017) |
How to design experiments assessing crosstalk between S401 phosphorylation and other post-translational modifications?
Methodological Answer
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Co-Immunoprecipitation (Co-IP): Use antibodies against acetylation (e.g., K389/K390 ) or SUMOylation sites alongside phospho-S401 antibodies.
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Kinase/Phosphatase Inhibition: Treat cells with PI3K/Akt inhibitors (LY294002) or MAPK activators to dissect signaling hierarchies .
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Mass Spectrometry: Perform LC-MS/MS on immunoprecipitated GATA2 to map coexisting modifications .
Key Finding: In AML, hyperphosphorylation at S401 (via Ras-MAPK) synergizes with acetylation at K389/K390 to amplify pro-leukemic gene expression (e.g., CXCL2) .
What strategies mitigate cross-reactivity in non-human models?
Methodological Answer
While most antibodies target human/monkey epitopes , use:
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Epitope Alignment: Compare S401 flanking sequences across species (e.g., mouse: K-M-S-N-K vs. human: K-M-SP-N-K) .
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CRISPR Mutagenesis: Introduce humanized S401 sequences into murine models.
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Alternate Assays: If cross-reactivity fails, switch to ELISA with human-specific capture antibodies .
Data Insight: The Invitrogen PA537583 antibody shows 10-fold lower affinity for murine phospho-GATA2 due to a single residue divergence .
How to integrate phospho-GATA2 (S401) data with transcriptomic/proteomic datasets?
Methodological Answer
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Pathway Enrichment: Use tools like GSEA to link phospho-GATA2 levels to:
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Multi-Omics Correlation: Overlay phosphoproteomics data with RNA-seq from the same samples (e.g., TCGA cohorts ).
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Machine Learning: Train classifiers to predict phosphorylation status using upstream kinase activity (Akt, MAPK) .
Case Study: In prostate cancer, phospho-GATA2 (S401) correlates with ST6GALNAC5 expression (AUC = 0.93 for metastasis prediction) .
How to troubleshoot inconsistent phosphorylation signals in time-course experiments?
Solution Workflow:
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Kinetics Optimization: Sample every 2–5 min post-stimulation (e.g., TNFα peaks at 5 min , insulin at 15 min ).
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Proteostasis Control: Inhibit proteasomes (10 μM MG132) to stabilize transient phosphorylation .
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Signal Normalization: Express data as a ratio of phospho-GATA2/total GATA2 .
What controls are essential for ChIP-seq using phospho-GATA2 (S401) antibodies?
Required Controls:
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Non-phospho Competitor: Add 10x molar excess of non-phosphorylated peptide .
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Site-Directed Mutants: Compare DNA binding in S401A vs. S401D mutants .
Pitfall: Phospho-specific antibodies may reduce ChIP efficiency due to epitope masking; optimize crosslinking time (8–12 min) .
Can phospho-GATA2 (S401) status predict therapeutic response in hematologic malignancies?
Evidence-Based Approach:
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Biomarker Profiling: In MDS/AML patients, quantify phospho-GATA2 levels via ELISA and correlate with:
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PDX Models: Treat patient-derived xenografts with Akt inhibitors and monitor phospho-GATA2 dynamics .
Table 3: Clinical Correlations in GATA2 Dysregulation
| Condition | Phospho-S401 Level | Therapeutic Implication |
|---|---|---|
| MonoMAC syndrome | Elevated | Resistant to IFN-γ therapy |
| Obesity-linked insulin resistance | Reduced | Sensitive to PPARγ agonists |
How to investigate evolutionary conservation of S401 phosphorylation?
Phylogenetic Methodology:
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Sequence Alignment: Use Clustal Omega to align GATA2 across 50+ species.
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In Silico Prediction**: Scan for Akt/PI3K recognition motifs (RXRXXS/T) near S401.
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Functional Assays: Express zebrafish/human chimeric GATA2 in Xenopus models and assess erythropoiesis .
Finding: The S401 motif is conserved in primates and rodents but absent in teleosts, suggesting a mammalian-specific regulatory role .
