Phospho-TCF3 (T355) Antibody
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Product Specs
Buffer
Liquid in PBS containing 50% glycerol, 0.5% BSA and 0.02% sodium azide.
Form
Liquid
Lead Time
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Synonyms
AGM8 antibody; bHLHb21 antibody; Class B basic helix-loop-helix protein 21 antibody; E12 antibody; E2A antibody; E2A immunoglobulin enhancer binding factors E12/E47 antibody; E47 antibody; Helix loop helix protein HE47 antibody; Immunoglobulin enhancer-binding factor E12/E47 antibody; Immunoglobulin transcription factor 1 antibody; ITF1 antibody; Kappa-E2-binding factor antibody; MGC129647 antibody; MGC129648 antibody; Negative vitamin D response element binding protein antibody; NOL1-TCF3 fusion antibody; TCF-3 antibody; Tcf3 antibody; TFE2_HUMAN antibody; transcription factor 3 (E2A immunoglobulin enhancer binding factors E12/E47) antibody; Transcription factor 3 antibody; transcription factor 3 variant 3 antibody; Transcription factor E2-alpha antibody; Transcription factor ITF-1 antibody; VDIR antibody; VDR interacting repressor antibody; vitamin D receptor-interacting repressor antibody
Target Names
Uniprot No.
Target Background
Function
TCF3 (also known as E2A) is a transcriptional regulator that plays a crucial role in the initiation of neuronal differentiation and mesenchymal to epithelial transition. Heterodimers formed between TCF3 and tissue-specific basic helix-loop-helix (bHLH) proteins are essential in determining tissue-specific cell fate during embryonic development, such as muscle or early B-cell differentiation. In collaboration with TCF15, TCF3 is required for the mesenchymal to epithelial transition. These dimers bind to DNA at E-box motifs with the sequence 5'-CANNTG-3'. TCF3 interacts with the kappa-E2 site within the kappa immunoglobulin gene enhancer, and binds to IEB1 and IEB2, short DNA sequences located in the insulin gene transcription control region.
Gene References Into Functions
- hnRNP H/F proteins are crucial for maintaining and differentiating embryonic stem cells. This function is partly attributed to a switch in TCF3 alternative splicing, which leads to repression of CDH1/E-cadherin. PMID: 30115631
- Patients with B-cell acute lymphoblastic leukemia exhibiting positive E2A-PBX1 fusion expression after transplant have a poor prognosis. PMID: 29705861
- Studies have shown that abnormal expression of endometrial E2A occurs in the mid-secretory endometrium of women experiencing recurrent miscarriage. Additionally, a positive correlation exists between E2A and FOXP3, and E2A and CTLA-4, suggesting a potential regulatory role of E2A in endometrial receptivity. PMID: 29270752
- Silencing of the TCF3 gene inhibits esophageal cancer cell growth and proliferation, suppresses cell cycle progression, and promotes apoptosis. PMID: 28864779
- Research suggests that the upregulation of TCF3 is a critical prognostic factor for nasopharyngeal carcinoma. PMID: 28107608
- Findings indicate that TCF3 is clearly associated with the progression of cervical squamous cell carcinoma. This is the first report demonstrating that TCF3 can act as a tumor promoter in cervical cancer, potentially having significant implications for CSCC prognosis. PMID: 28604457
- Inactivation of TCF3 contributes to the oncogenic program of classical Hodgkin lymphoma. PMID: 27166193
- This study represents the first identification of protein partners for both E2A-PBX1 and HOXA9 oncoproteins through an unbiased biochemical approach. The identification of translation initiation factors associated with HOXA9 suggests a novel function for HOX proteins independent of their transcriptional activity. PMID: 28707666
- Poly (ADP-ribose) polymerase inhibitors selectively induce cytotoxicity in TCF3-HLF-positive leukemic cells. PMID: 27894958
- MiR-138 may function as a tumor suppressor and a potential prognostic biomarker in cervical cancer. Its downstream target, TCF3, may also regulate cancer development in a reverse manner as miR-138. PMID: 28385388
- High levels of TCF3 in gliomas promote glioma development through the Akt and Erk pathways. PMID: 27105323
- TWIST1-E12 protein heterodimeric complexes may constitute the main active forms of TWIST1 in relation to senescence inhibition during the course of breast tumorigenesis. PMID: 27237323
- A comprehensive review of the role of the E2A-PBX1 gene rearrangement in the prognosis of childhood acute lymphoblastic leukemia and its central nervous system relapse. PMID: 26509298
- E47 is a novel substrate of PAK5, and PAK5-mediated phosphorylation of E47 promotes epithelial-mesenchymal transition. High expression of phospho-E47 is associated with an aggressive phenotype of colon cancer and metastasis. PMID: 26212009
- Significant enrichment of the neuroactive ligand-receptor interaction pathway was observed in TCF3-PBX1, as well as enrichment of genes involved in immunity and infection pathways in the ETV6-RUNX1 subtype. PMID: 26237075
- Overexpression of E47 reprograms human pancreatic cancer cells to a quiescent acinar state with reduced tumorigenic potential. PMID: 25894862
- Drug response profiling of matched patient-derived xenografts revealed a distinct profile for TCF3-HLF ALL with resistance to conventional chemotherapeutics but sensitivity to glucocorticoids. PMID: 26214592
- When intensive chemotherapy was employed, TCF3-PBX1 was associated with a favorable outcome in childhood pre-B ALL. PMID: 25551271
- Inhibitor of differentiation 4 (ID4) acts as an inhibitor of ID-1, -2 and -3, promoting basic helix loop helix (bHLH) E47 DNA binding and transcriptional activity. PMID: 25778840
- Data suggests that microRNAs miR-155 and miR-16 downregulate activation-induced cytidine deaminase (AID) and transcription factor E47 in B Cells through binding of the 3'-untranslated regions. PMID: 26223652
- TCF3 is a novel susceptibility locus for Hodgkin lymphoma. PMID: 24920014
- Research suggests a collaborative action between autophagy and ubiquitination in the degradation of E2A/Pbx1, revealing a novel strategy for targeted preventive or treatment therapy in pediatric ALL. PMID: 25615280
- TCF3 rearrangement [t(1;19) (q23;p13)] was detected in 16 (20%) of the 80 studied patients and was significantly associated with splenomegaly, lymphadenopathy, and CNS infiltration. PMID: 25116187
- Results indicate that the up-regulation of TCF3, primarily caused by promoter hypomethylation, is one of the molecular mechanisms involved in the development and progression of colorectal cancer. PMID: 25375219
- Results suggest that E47 has diverse effects in T-ALL, but functional deficiency of E47 is not a universal feature of Lmo2-induced T-ALL. PMID: 25499232
- In this study, we demonstrated that TCF3-PBX1 positive pediatric BCP-ALL patients treated according to the JACLS ALL02 and CCLSG ALL2004 protocol had favorable outcomes. PMID: 24578304
- Results suggest that E2A suppresses CRC cell metastasis, at least partially if not all, by inhibiting YAP expression. PMID: 24369055
- E2A is an independent prognostic factor for colorectal cancer patients and targets miR-320a to regulate cell proliferation of colon cancer cells. PMID: 24454819
- Within the AETFC complex, AML1-ETO oligomerization is required for a specific interaction between the oligomerized NHR2 domain and a novel NHR2-binding (N2B) motif in E proteins, including HEB and E2A. Disruption of this interaction by point mutations abrogates AML1-ETO-induced hematopoietic stem/progenitor cell self-renewal and leukemogenesis. PMID: 23812588
- In t(8;21) leukemia cells, the two E proteins, HEB and E2A, function as components of the stable AML1-ETO-containing transcription factor complex (AETFC). The AETFC components cooperatively regulate gene expression and contribute to leukemogenesis. PMID: 23812588
- NF-kappaB potentiated the binding of E2A to an E-box motif located immediately downstream of the 2 closely-spaced transcription start sites for sustained 14-3-3gamma expression and CSR induction. PMID: 23851690
- Four patients with agammaglobulinemia exhibited the same de novo mutation in the broadly expressed transcription factor E47. The mutant protein (E555K) was stable in patient-derived EBV-transformed cell lines and cell lines transfected with expression vectors. PMID: 24216514
- E47 interacts with Id1 in E47 overexpressing MDCK cells that underwent a full epithelial-mesenchymal transition as well as in mesenchymal breast carcinoma and melanoma cell lines. PMID: 23555842
- The E2A-PBX1 fusion gene caused by t(1;19)(q23;p13) may be a common genetic change in AIS and a survival determinant for female AIS patients at an early stage. PMID: 23688269
- High expression of TCF3 is associated with neuroblastoma progression. PMID: 23135478
- This study provides the first evidence that the portion of E2A proteins found within the oncogenic fusion protein E2A-PBX1, including the transcriptional activation domains, can enhance the HAT activity of CBP/p300. PMID: 22387215
- Findings demonstrate that E2A proteins are acetylated at specific residues as a consequence of lysine acetyltranferases (CBP, p300, and PCAF) recruitment. PMID: 22207202
- The molecular mechanism underlying the effect of EB1089 on aromatase gene expression was found to be mediated by the vitamin D receptor (VDR), vitamin D receptor interacting repressor (VDIR), and Williams syndrome transcription factor (WSTF). PMID: 23085504
- E2A-HLF sensitizes t(17;19)-positive acute lymphoblastic leukemia to graft-versus-leukemia effect by upregulating death receptors for TRAIL. PMID: 22743623
- The absence of T cell precursor potential, both in vivo and in vitro, is attributed to low Notch1 expression and secondary to inhibition of E2A protein activity by members of the inhibitor of DNA binding (Id) protein family. PMID: 22972921
- Protein inhibitor of activated STAT, PIASy regulates alpha-smooth muscle actin expression by interacting with E12 in mesangial cells. PMID: 22829926
- These results demonstrate for the first time that E2A could indeed act as a tumor promoter, at least in prostate cancer. PMID: 22564737
- Studies have shown that the entire ensemble of Id proteins has the ability to interact with E47, identify factors that associate with E47, and reveal a spectrum of phosphorylated residues in E47, including an AKT substrate site. PMID: 22354994
- The E2A, a key transcription factor associated with the B-cell activation profile, regulates apoptosis in CLL and may contribute to disease pathology. PMID: 21551245
- Altering the balance between Id3 and E47 is both necessary and sufficient to regulate the cell cycle in PDA cells. PMID: 21498546
- E2A exhibits a tumor suppressor function in human lymphoid cells. PMID: 21788410
- Results indicate that in cancer cells, E2A, FOXO1, and FOXP1 regulate RAG1 and RAG2 expression, initiating Ig gene rearrangement in a manner similar to B lymphocytes. PMID: 21655267
- HEB and E2A bind to the SCA motif at regions overlapping SMAD2/3 and FOXH1. PMID: 21828274
Database Links
Involvement In Disease
Agammaglobulinemia 8, autosomal dominant (AGM8)
Subcellular Location
Nucleus.
Q&A
How can researchers validate the specificity of Phospho-TCF3 (Thr355) antibodies in heterogeneous tissue samples?
Validation of antibody specificity requires a multi-pronged approach:
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Knockout/knockdown controls: Use cell lines or tissues lacking TCF3 or with CRISPR-mediated Thr355 mutations to confirm loss of signal . For example, studies in TCF3 haploinsufficiency models showed reduced antibody reactivity in heterozygous B cells compared to wildtype .
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Peptide competition assays: Pre-incubate the antibody with phosphorylated Thr355 peptides to block binding. A ≥80% reduction in signal confirms specificity .
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Cross-species validation: Test reactivity in human, mouse, and rat samples, as TCF3 Thr355 is conserved across these species .
Key data:
| Validation Method | Expected Outcome | Reference |
|---|---|---|
| TCF3-null B cells | No detectable signal | |
| Phosphopeptide blocking | Signal inhibition >80% | |
| Multi-species IHC | Consistent staining patterns |
What experimental conditions optimize Phospho-TCF3 (Thr355) detection in Western blotting?
Optimal conditions depend on phosphorylation dynamics and tissue type:
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Lysis buffers: Include phosphatase inhibitors (e.g., 1 mM Na3VO4, 10 mM β-glycerophosphate) to preserve phosphorylation .
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Gel electrophoresis: Use 4–12% Bis-Tris gels to resolve TCF3 isoforms (E12: ~65 kDa; E47: ~75 kDa) .
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Blocking agents: 5% BSA in TBST reduces non-specific binding compared to skim milk .
Critical parameters:
How does Thr355 phosphorylation modulate TCF3’s transcriptional activity in B-cell development?
Phosphorylation at Thr355 alters TCF3’s dimerization capacity and DNA binding:
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Mechanism: Thr355 resides in the activation domain, influencing interactions with co-factors like E proteins and ID inhibitors . Mutations at this site (e.g., T355E phosphomimetic) reduce binding to ID3, enhancing E47 homodimer formation .
-
Functional impact: In Burkitt lymphoma, Thr355-phosphorylated TCF3 drives CCND3 and PTPN6 overexpression, promoting cell cycle progression .
Supporting data:
| Model System | Finding | Source |
|---|---|---|
| BL cell lines | TCF3 Thr355 mutants increase E47:TCF3 ratio by 2.5x | |
| Murine B cells | TCF3 Thr355Ala reduces IgM+ cells by 40% |
What are common pitfalls in interpreting Phospho-TCF3 (Thr355) staining in immunohistochemistry (IHC)?
Challenges include cross-reactivity and epitope masking:
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False positives: Endogenous biotin in liver/kidney samples may require streptavidin-biotin blocking .
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Epitope accessibility: Antigen retrieval with citrate buffer (pH 6.0, 95°C, 20 min) improves detection in FFPE tissues .
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Quantitative limits: Semi-quantitative scoring (H-score) is preferable to binary +/- calls due to variable phosphorylation across cell cycles .
Troubleshooting table:
| Issue | Solution |
|---|---|
| Weak signal | Increase primary antibody incubation (24 hrs at 4°C) |
| High background | Optimize peroxidase quenching (0.3% H2O2, 30 min) |
| Nuclear/cytoplasmic mismatch | Validate with subcellular fractionation |
How do TCF3 phosphorylation states correlate with clinical phenotypes in immunodeficiency studies?
Phospho-TCF3 levels are biomarkers for immune dysregulation:
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Haploinsufficiency: Monoallelic TCF3 LOF reduces Thr355 phosphorylation by 60%, correlating with hypogammaglobulinemia (IgG <4 g/L) .
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T-cell signaling: In Tregs, Thr355 phosphorylation peaks at 5 min post-TCR stimulation, 2x faster than in non-Tregs .
Clinical correlations:
| Patient Cohort | Phospho-TCF3 Level | Clinical Feature |
|---|---|---|
| TCF3 Null | Undetectable | Agammaglobulinemia |
| TCF3 E555K (DN) | Reduced 70% | Recurrent pneumonia |
| BL-associated mutants | Elevated 3x | CCND3-driven proliferation |
What orthogonal methods confirm Phospho-TCF3 (Thr355) antibody specificity in single-cell assays?
Combine antibody-based detection with functional readouts:
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Flow cytometry: Pair with intracellular staining for downstream targets (e.g., CD19 in B cells) .
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Proximity ligation assays (PLA): Validate phosphorylation-dependent TCF3-ID3 interactions .
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Mass spectrometry: Verify Thr355 phosphorylation in immunoprecipitated TCF3 .
Validation workflow:
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Sort CD19+ B cells using magnetic beads.
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Stain with Phospho-TCF3 (Thr355) antibody and anti-CD19-PE.
How do somatic TCF3 Thr355 mutations in Burkitt lymphoma alter splicing regulation?
Recurrent mutations (e.g., T1670A, G1681A) dysregulate hnRNPH1 binding:
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Splicing impact: Mutations in exon 18b reduce hnRNPH1 affinity by 30%, increasing E47 isoform production .
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Functional consequence: Elevated E47 upregulates PTPN6 (2.8x) and CCND3 (1.9x), driving lymphomagenesis .
Mechanistic insights:
| Mutation | Effect on Splicing | Target Gene Dysregulation |
|---|---|---|
| T1670A | Exon 18b inclusion ↑40% | PTPN6 ↑2.5x |
| G1681A | hnRNPH1 binding ↓35% | CCND3 ↑1.7x |
What in vivo models best recapitulate human TCF3 phosphorylation dynamics?
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Murine models: Tcf3 haploinsufficient mice show 50% reduced B220+ cells but normal Treg function, diverging from human phenotypes .
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Xenografts: BL patient-derived xenografts (PDX) retain Thr355 phosphorylation patterns for ≥4 passages .
Model comparison:
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