TFAP2B Antibody

What is TFAP2B and what are its key structural and functional characteristics?

TFAP2B is a nuclear transcription factor that binds to the consensus sequence 5'-GCCNNNGGC-3' to regulate genes involved in development, differentiation, and various physiological processes. In humans, the canonical protein:

• Contains 460 amino acid residues with a molecular weight of 50.5 kDa

• Has predominant nuclear localization

• Exists in up to 2 different isoforms

• Undergoes post-translational modifications, including sumoylation

TFAP2B plays critical roles in:

• Development of eye, face, body wall, limb, and neural tube structures

• Fat cell differentiation and carbohydrate metabolism

• Suppression of genes including MCAM/MUC18, C/EBP alpha, and MYC

• Terminal differentiation and function of renal tubular epithelia

What are the validated applications for TFAP2B antibodies in research?

TFAP2B antibodies have been successfully employed in multiple experimental applications:

For optimal results in immunohistochemistry, 4% paraformaldehyde fixation followed by antigen retrieval (Target Retrieval Solution at pH 9, boiled at 108°C for 15 minutes) has been validated in multiple studies .

How is TFAP2B expression distributed in normal and pathological tissues?

TFAP2B shows distinct expression patterns across various tissues:

Normal Tissues:

• Expressed in the cerebellum, where it marks specific neuronal populations including Cerebellar Inhibitory Neurons, Midbrain Splatter Neurons, and Amacrine Cells

• In breast tissue, serves as a luminal mammary epithelial differentiation marker in GATA3-positive cells

• Co-expression with steroid receptors: 78% of AR-positive lobular cells and 76% of ductal cells express TFAP2B; 67% of ER-positive lobular cells and 62% of ductal cells express TFAP2B

Pathological Tissues:

• Overexpressed in lung adenocarcinomas, correlating with poor prognosis (P < 0.001)

• Elevated in thyroid cancer tissues compared to adjacent normal tissues

• Expression levels altered in neuroblastoma, with low TFAP2B expression associated with poor outcome

What role does TFAP2B play in neuronal development and differentiation?

TFAP2B is essential for proper neuronal development, particularly in the cerebellum:

• Expression sequence: Follows Ptf1a and Olig2 (GABAergic fate determinants) and precedes Pax2 (interneuron marker)

• Selective expression: While TFAP2A is expressed by all GABAergic neurons, TFAP2B is selectively expressed by interneurons

• Functional significance: In utero electroporation (IUE) experiments demonstrate that:

  • TFAP2B knockdown using shRNA (pU6-2Bsh2) reduces TFAP2B expression by approximately 20-fold

  • TFAP2B overexpression increases expression by approximately 5-fold

• In neuroblastoma, TFAP2B mediates noradrenergic neuronal differentiation in vitro

When investigating TFAP2B in neuronal contexts, researchers should consider co-staining with appropriate neuronal markers (MAP2, TUBB3) to accurately characterize TFAP2B-expressing populations .

How can researchers effectively optimize TFAP2B knockdown and overexpression studies?

For successful genetic manipulation of TFAP2B:

Knockdown Strategies:

• siRNA approach: Test multiple sequences to identify most efficient (e.g., pU6-2Bsh2 demonstrated high specificity)

• Vector design: Effective constructs include short hairpin RNA under mouse U6 promoter with GFP reporter (pU6-shRNA-CMV-GFP)

• Validation: Western blot confirmation of knockdown efficiency is essential

Overexpression Strategies:

• Expression vector: Effective constructs include cDNA expressing TFAP2B with fluorescent reporter (pEF1-cDNA-IRES-RFP)

• Cell lines: HEK-293T cells (lacking endogenous TFAP2B) are suitable for initial validation

In Vivo Delivery Methods:

• In utero electroporation (IUE): Successfully used to deliver constructs to fourth ventricle of E12.5 embryos

• Assessment timeframe: Transfected neurons can be analyzed after 3 days as they migrate into the white matter

For functional readouts, researchers should assess:

• Cell proliferation (MTT assay)

• Colony formation capacity

• Cell morphology (spreading, filopodia formation)

• Apoptosis markers (caspase activation)

What are the molecular mechanisms by which TFAP2B influences cancer progression?

TFAP2B contributes to cancer development through multiple mechanisms:

Signaling Pathway Modulation:

• ERK/p38 pathway: TFAP2B knockdown inhibits this proliferation-associated pathway in lung cancer

• Caspase/cytochrome-c pathway: TFAP2B affects apoptotic signaling

• VEGF/PEDF-dependent pathway: TFAP2B regulates angiogenesis factors

Transcriptional Regulation:

• COX-2 activation: TFAP2B binds directly to the COX-2 promoter in thyroid cancer cells

  • Confirmed by biotin-labeled promoter pulldown and luciferase reporter assays

  • TFAP2B knockdown decreases COX-2 promoter activity

  • TFAP2B overexpression increases COX-2 expression at both protein and RNA levels

Phenotypic Effects:

• Cell morphology: TFAP2B knockdown reduces cell-to-cell contact and filopodia formation

• Colony formation: TFAP2B knockdown significantly decreases both colony formation ratio and colony size in soft agar assays

• Tumor growth: In xenograft models, TFAP2B knockdown inhibits tumor development and reduces angiogenic markers

How does TFAP2B regulate BMP signaling during development?

TFAP2B exhibits complex regulation of Bone Morphogenetic Protein (BMP) signaling:

Promoter Binding:

• In silico analysis identified multiple TFAP2 binding sites in BMP2 and BMP4 promoter regions

• Gel shift assays confirmed that specific sequences from both promoters bind TFAP2 proteins with different affinities

Differential Regulation:

• BMP2: TFAP2B activates the BMP2 promoter, increasing activity 3-6 fold in a dose-dependent manner (P<0.01)

• BMP4: TFAP2B represses the BMP4 promoter, reducing activity 2.5-4 fold (P<0.01)

Developmental Significance:

• Tfap2b-deficient mice display heart-hand defects similar to human Char syndrome (caused by TFAP2B mutations)

• Altered BMP2/BMP4 expression ratio likely contributes to these developmental abnormalities

This regulatory mechanism demonstrates how TFAP2B orchestrates the balance of BMP signaling crucial for proper cardiovascular and limb development.

What technical considerations are important when detecting TFAP2B in difficult experimental contexts?

Researchers face several challenges when working with TFAP2B:

Nuclear Protein Extraction:

• TFAP2B is predominantly nuclear, requiring optimized nuclear extraction protocols

• Minimize cross-contamination between nuclear and cytoplasmic fractions

• Consider specialized buffers with protease inhibitors to preserve integrity

Antigen Retrieval for Fixed Tissues:

• TFAP2B epitopes may be masked by fixation

• Validated protocol: Target Retrieval Solution (pH 9) with high-temperature treatment (108°C for 15 minutes)

• Include positive controls (cerebellum, breast tissue) and negative controls (primary antibody replaced with normal IgG)

Co-expression Analysis:

• For tissues with heterogeneous cell populations, co-staining is essential

• In breast tissue, double-immunofluorescence staining has been effective for evaluating TFAP2B co-expression with hormone receptors

• For cerebellar studies, neuronal subtype markers help identify specific TFAP2B-expressing populations

Quantification Methods:

• For IHC quantification, integral optical density (IOD) measurement using software like Image Plus Pro provides objective assessment

• When comparing expression levels across multiple samples, standardized acquisition settings and calibration controls are critical

How can researchers investigate TFAP2B DNA-binding properties and target gene identification?

To study TFAP2B interaction with genomic DNA:

Binding Site Identification:

• Electrophoretic Mobility Shift Assays (EMSAs): Design oligonucleotides containing putative binding sites (consensus 5'-GCCNNNGGC-3')

• Competition assays with unlabeled oligonucleotides and supershift assays with anti-TFAP2B antibodies confirm specificity

• ChIP-seq provides genome-wide identification of binding sites

Functional Validation:

• Luciferase reporter assays: Subclone promoter fragments into reporter vectors

  • Successful examples include 2225bp BMP2 promoter and 1618bp BMP4 promoter fragments

• Site-directed mutagenesis of binding sites can confirm their functional relevance

Bioinformatic Analysis:

• ConSites service and JASPAR datasets improve prediction accuracy

• Consider evolutionary conservation of binding sites across species

• Note that computational predictions have high sensitivity but low selectivity—experimental validation is essential

Expression Correlation:

• In thyroid cancer, TFAP2B expression positively correlates with COX-2 expression (P<0.05)

• When studying novel target genes, correlative expression analysis between TFAP2B and candidate targets provides supporting evidence

What is the significance of TFAP2B in Char Syndrome and other developmental disorders?

TFAP2B mutations cause Char syndrome, characterized by:

Clinical Features:

• Patent ductus arteriosus (PDA)

• Facial dysmorphism

• Hand anomalies

Molecular Pathology:

• TFAP2B regulates genes crucial for cardiovascular and skeletal development

• Altered BMP signaling: TFAP2B differentially regulates BMP2 (activation) and BMP4 (repression)

• Mouse models: Tfap2b-deficient mice recapitulate heart-hand defects similar to human Char syndrome

Research Applications:

• Patient-derived mutations can be studied using site-directed mutagenesis

• Functional assays to assess mutant TFAP2B include:

  • DNA binding capacity

  • Transcriptional activity on target gene promoters

  • Protein stability and subcellular localization

Understanding the molecular mechanisms of TFAP2B in development provides insights not only for Char syndrome but also for more common conditions involving patent ductus arteriosus and limb abnormalities.

How do TFAP2B expression patterns differ across model organisms?

TFAP2B shows evolutionary conservation with species-specific variations:

Cross-Species Conservation:

• TFAP2B orthologs reported in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken species

• Conservation of key functional domains, particularly DNA-binding regions

Species-Specific Functions:

• Mouse: Essential for GABAergic interneuron development in cerebellum

• Zebrafish: Contributes to neural crest development and craniofacial morphogenesis

• Human: Mutations cause Char syndrome with characteristic facial, cardiac, and limb abnormalities

Antibody Cross-Reactivity:

• Many commercial antibodies raised against human TFAP2B cross-react with mouse and rat orthologs

• When working with less common model organisms, antibody validation is essential

Experimental Considerations:

• For interspecies comparisons of TFAP2B function, consider differences in:

  • Developmental timing of expression

  • Tissue distribution

  • Regulatory networks

  • Target gene repertoire

Understanding these cross-species similarities and differences is crucial for translating findings between model organisms and human disease.

What methods are optimal for studying TFAP2B post-translational modifications?

TFAP2B undergoes several post-translational modifications that affect its function:

Sumoylation:

• TFAP2B is known to undergo sumoylation, which can affect its transcriptional activity

• Detection methods include:

  • Immunoprecipitation followed by western blotting with anti-SUMO antibodies

  • Use of SUMO-specific proteases to confirm modification

Other Potential Modifications:

• Phosphorylation sites may regulate TFAP2B activity in response to signaling pathways

• Acetylation could affect DNA binding or protein-protein interactions

Experimental Approaches:

• Mass spectrometry analysis of immunoprecipitated TFAP2B can identify modification sites

• Mutagenesis of putative modification sites followed by functional assays

• Pharmacological inhibitors of modification pathways can reveal regulatory mechanisms

Functional Significance:

• Post-translational modifications likely contribute to:

  • Context-dependent gene regulation

  • Integration of TFAP2B with cellular signaling networks

  • Differential activity during development and disease progression

Understanding these modifications provides deeper insight into the mechanisms controlling TFAP2B function in various biological contexts.