TBX4 Antibody

What cellular localization pattern should be expected when using TBX4 antibodies in immunofluorescence studies?

TBX4 is primarily localized in the nucleus, where it functions as a transcription factor binding to specific DNA sequences to regulate target gene expression. Immunofluorescence studies have demonstrated that both wild-type and mutant TBX4 proteins are evenly expressed in the nucleus, suggesting that most mutations do not affect nuclear translocation . When performing immunofluorescence with TBX4 antibodies, researchers should expect a distinct nuclear staining pattern and can use this localization as a quality control measure for antibody specificity.

What are the most reliable detection methods when working with TBX4 antibodies?

TBX4 antibodies can be detected through multiple methods including:

• Western blotting (WB): Effective for detecting TBX4 protein expression levels and molecular weight

• Immunoprecipitation (IP): Useful for protein-protein interaction studies involving TBX4

• Immunofluorescence (IF): Ideal for visualizing cellular localization of TBX4

• Enzyme-linked immunosorbent assay (ELISA): Suitable for quantitative analysis

For optimal results, mouse monoclonal antibodies such as TBX4 Antibody (G-4) have demonstrated reliable detection of human TBX4 protein across these methods . When designing experiments, consider that TBX4 antibodies are available in various conjugated forms (including agarose, HRP, PE, FITC, and Alexa Fluor conjugates) to accommodate different experimental requirements .

What cell types are most appropriate for studying TBX4 expression and function?

Based on current research, the following cell types are most appropriate for TBX4 studies:

• Pulmonary fibroblasts (particularly fetal lung fibroblasts)

• Pulmonary artery smooth muscle cells (PASMCs)

• Pulmonary endothelial cells

• Mesenchymal stem cells (MSCs)

MSCs have been successfully used to study TBX4 mutations and represent a valuable model system for investigating TBX4 function . These cells can be transfected with wild-type or mutant TBX4 constructs and maintained with stable expression, making them ideal for comparative functional studies .

How can I optimize chromatin immunoprecipitation (ChIP) protocols when using TBX4 antibodies?

ChIP optimization for TBX4 antibodies requires several critical considerations:

• Crosslinking conditions: Standard formaldehyde fixation (1%) for 10 minutes at room temperature has been successfully used in TBX4 ChIP experiments.

• Sonication parameters: Aim for DNA fragments between 200-500bp for optimal TBX4 binding site resolution.

• Antibody validation: Pre-validate TBX4 antibody specificity using Western blotting before ChIP applications.

• Control primers: Include GAPDH as a negative control region to verify specificity of TBX4 binding .

• Target region selection: When investigating TBX4 autoregulation, design primers targeting the TBX4 proximal promoter region (-558 to -1) which contains TBX binding motifs and Smad binding elements .

• Quantification method: ChIP-qPCR has been successfully employed to measure TBX4 binding to promoter regions of target genes like FGF10 .

Research has demonstrated that ChIP can effectively detect TBX4 binding to its own promoter, suggesting autoregulation, and to promoters of target genes involved in development and fibrosis .

What are the key considerations when investigating TBX4 mutations and their functional effects?

When investigating TBX4 mutations:

• Domain focus: Prioritize the T-box domain (amino acids 71-251) as approximately 75% of missense mutations occur within this crucial DNA-binding region .

• Functional assays:

  • Dual luciferase reporter assays to measure transcriptional activity

  • ChIP to assess DNA binding capacity

  • Western blot analysis to evaluate effects on downstream signaling (particularly p-Smad1/5 levels)

• Target gene expression: Measure expression of known TBX4 targets such as FGF10, as reduced expression has been observed with TBX4 mutations .

• Binding vs. activation distinction: TBX4 mutations may still permit binding to target promoters (as detected by ChIP) while significantly reducing transcriptional activation (as measured by luciferase assays) , emphasizing the importance of functional validation beyond binding studies.

• Signaling pathway analysis: Investigate effects on BMP-Smad signaling, as TBX4 mutations have been shown to reduce p-Smad1/5 levels in patient-derived cells .

How can I design experiments to investigate the role of TBX4 in fibrotic processes?

To investigate TBX4's role in fibrosis:

• Lineage tracing: Utilize Tbx4-Cre or LME-Cre Rosa26-tdTomato mouse models to track Tbx4-lineage cells during fibrotic responses .

• Fibrosis induction: The bleomycin lung injury model has been effective for studying TBX4's role in pulmonary fibrosis, with analysis at 21 days post-injury showing clear overlap between tdT+ (Tbx4-lineage) cells and fibroblast markers .

• Marker co-localization: Analyze co-expression of:

  • αSMA (myofibroblast marker)

  • Collagen type 1 α1 (COL1α1)

  • Desmin and vimentin (mesenchymal markers)

  • PDGFRβ and NG2 (pericyte markers)

• Loss-of-function approaches: CRISPR-Cas9-mediated knockout of TBX4 can be used to assess its necessity in fibrotic processes .

• Gain-of-function studies: Overexpression of TBX4 to determine if it accelerates fibrotic responses .

• Signaling pathway analysis: Investigate p-Smad1/5 levels as TBX4 modulates this pathway, which could be a mechanism linking TBX4 to fibrotic processes .

What are the methodological approaches for studying TBX4-mediated transcriptional regulation?

To investigate TBX4's transcriptional regulatory functions:

• Promoter analysis: Scan target gene promoters for TBX binding motifs, Smad binding elements, and BMP response elements (5GC sites) . For example, the TBX4 proximal promoter region (-558 to -1) contains these regulatory elements .

• Reporter constructs: Generate luciferase reporter constructs containing target promoters (e.g., pGL4.23-TBX4 or pGL3-FGF10) .

• Dose-response experiments: Co-transfect cells with target promoter reporters and increasing doses of TBX4 expression vectors to establish dose-dependent regulation .

• Domain function analysis: Compare full-length TBX4 with domain deletion mutants (e.g., TBX4ΔT-Box) to identify essential domains for transcriptional activity .

• ChIP-qPCR: Quantify binding of TBX4 and co-factors (like p-Smad1/5) to target promoters .

• Downstream target analysis: Measure expression of established TBX4 targets such as FGF10 and ID1/2/3 genes using qRT-PCR after TBX4 manipulation .

How can I assess the relationship between TBX4 and BMP-Smad signaling in pulmonary arterial hypertension models?

To investigate TBX4-BMP-Smad interactions in PAH:

• Cell models:

  • Patient-derived lymphocyte cell lines carrying TBX4 mutations (such as frameshift deletions)

  • Pulmonary artery smooth muscle cells (PASMCs)

  • Pulmonary endothelial cells

• Signaling analysis:

  • Western blot for p-Smad1/5 levels after TBX4 overexpression or knockdown

  • qRT-PCR for downstream targets (ID1, ID2, ID3)

• Induction experiments: Treat cells with BMP4 to assess effects on TBX4 expression levels and determine if TBX4 is a target of Smad signaling .

• Feedback loop investigation: Analyze the positive feedback relationship between TBX4 and p-Smad1/5 using both overexpression and knockdown approaches .

• Mutational analysis: Compare wild-type TBX4 with PAH-associated mutations, particularly those in the T-box domain (aa 71-251) .

• Target gene regulation: Assess whether TBX4 mutations alter regulation of genes involved in vascular remodeling, which could explain the PAH phenotype .

What are common issues when performing immunofluorescence with TBX4 antibodies and how can they be resolved?

Common challenges and solutions:

• High background signal:

  • Increase blocking time (use 5% normal serum for at least 1 hour)

  • Optimize primary antibody dilution (typically 1:200 to 1:500 works well for TBX4 antibodies)

  • Extend washing steps (at least 3 washes of 5 minutes each)

• Weak or absent nuclear signal:

  • Ensure proper fixation (4% paraformaldehyde for 15 minutes)

  • Consider antigen retrieval methods if using paraffin sections

  • Verify antibody compatibility with your fixation method

• Non-specific cytoplasmic staining:

  • This may indicate antibody cross-reactivity or fixation issues

  • Validate antibody specificity using TBX4 knockdown/knockout controls

  • For TBX4, expect predominant nuclear localization as confirmed in multiple studies

• GFP-tagged TBX4 visualization:

  • When working with overexpressed GFP-tagged TBX4, fluorescence microscopy after 7 days of selection should show clear nuclear localization in most cells

  • Confirm expression with Western blot using GFP antibodies if fluorescence signal is weak

How can I interpret contradictory results between TBX4 binding to promoters versus functional activation?

When facing contradictory results:

• Binding without activation phenomenon:

  • TBX4 mutations may retain DNA binding capability (positive ChIP results) while showing impaired transcriptional activation (reduced luciferase activity)

  • This suggests TBX4's role may involve both DNA binding and recruitment of additional co-factors

• Resolution strategies:

  • Perform both ChIP and functional assays (luciferase) in parallel

  • For ChIP-positive but luciferase-negative results, investigate co-factor recruitment

  • Consider co-immunoprecipitation to identify missing interaction partners in mutant TBX4

• Control experiments:

  • Use multiple primer sets in ChIP to confirm binding specificity

  • Include domain deletion controls (e.g., TBX4ΔT-Box) that fail both binding and activation

• Quantitative assessment:

  • Evaluate binding efficiency quantitatively using ChIP-qPCR

  • Compare fold enrichment between wild-type and mutant TBX4

How might TBX4 antibodies be used to explore the relationship between lung development and fibrotic diseases?

TBX4 antibodies could advance this research through:

• Developmental stage analysis: Track TBX4-expressing cell populations during lung development and in response to injury using immunohistochemistry and lineage tracing .

• Cell fate mapping: Combine TBX4 antibodies with markers of epithelial-mesenchymal transition to investigate whether TBX4-positive cells contribute to fibrotic processes through cellular reprogramming.

• Single-cell applications: Use TBX4 antibodies for flow cytometry or single-cell sorting to isolate and characterize TBX4-expressing cells at different developmental stages and in pathological conditions.

• Spatial transcriptomics: Combine TBX4 immunolabeling with in situ transcriptomics to identify gene expression networks in TBX4-positive cells during development and fibrosis.

• Patient sample analysis: Apply TBX4 antibodies to patient-derived samples to correlate TBX4 expression patterns with clinical outcomes in fibrotic diseases and pulmonary arterial hypertension.

• Therapeutic target identification: Screen for compounds that modulate TBX4 function using antibody-based high-content imaging, potentially identifying agents that could interrupt fibrotic processes.

Current evidence indicates that TBX4 marks a population of cells that accumulate during lung fibrosis and express myofibroblast markers, suggesting developmental pathways may be reactivated in adult lung disease .