DLX4 Antibody

What is DLX4 and what biological functions does it serve?

DLX4, also known as BP1, DLX7, DLX8, or DLX9, is a member of the distal-less homeobox gene family. This protein plays several critical biological roles, including regulating the production of hemoglobin S and functioning as a transcriptional repressor. During embryonic development, DLX4 contributes significantly to palatogenesis and craniofacial development . DLX4 binds to two silencer DNA sequences upstream of the adult human β-globin gene and to a negative control region, suggesting its regulatory role in globin gene expression . Recent research has also implicated DLX4 in cancer development, with particular significance in tumor progression and immune cell recruitment .

What types of DLX4 antibodies are available for research applications?

Researchers have access to several types of DLX4 antibodies for experimental work:

• Polyclonal antibodies: These are typically produced in rabbits using recombinant DLX4 protein as the immunogen. Examples include the rabbit polyclonal antibody described in search result , which is purified through antigen affinity methods .

• Monoclonal antibodies: These offer high specificity with consistent results across experiments. The OTI7C8 clone is an example of a mouse monoclonal antibody against DLX4 .

• Recombinant monoclonal antibodies: These provide enhanced reproducibility and reduced batch-to-batch variation. The EPR9263 clone is an example of a rabbit recombinant monoclonal antibody against DLX4/BP1 .

Each antibody type offers distinct advantages depending on the experimental requirements and research questions being addressed.

Which experimental applications are supported by DLX4 antibodies?

DLX4 antibodies support multiple research applications, though validation status varies by manufacturer and clone:

Observed molecular weight by Western blot is approximately 38kDa for the polyclonal antibody , which differs from the predicted size of 26kDa reported for the recombinant monoclonal antibody . This discrepancy highlights the importance of validation in each experimental system.

How can DLX4 antibodies be utilized in cancer research?

DLX4 has emerged as a significant biomarker in cancer research, particularly in clear cell renal cell carcinoma (ccRCC). Studies have demonstrated that DLX4 expression correlates with several clinical parameters:

For cancer research applications, DLX4 antibodies are instrumental in:

• Immunohistochemical analysis of tumor tissue samples

• Western blot quantification of DLX4 protein expression in cancer cell lines

• Investigation of DLX4's role in tumor progression mechanisms

What molecular pathways and cellular processes are associated with DLX4 expression?

Functional enrichment analysis has revealed several pathways and cellular processes associated with DLX4 expression:

• Cell cycle regulation: DLX4 expression levels correlate with cell cycle-related pathways, suggesting a potential role in cellular proliferation .

• Epithelial-mesenchymal transition (EMT): This critical process in cancer metastasis shows association with DLX4 expression .

• Glycolysis: Metabolic reprogramming through glycolysis, a hallmark of cancer, is linked to DLX4 expression levels .

• Inflammatory response: DLX4 has been implicated in inflammatory processes that may contribute to cancer progression .

• Immune regulation: DLX4 may induce a megakaryocytic transcriptional program by activating IL-1β and NF-κB signaling. In ovarian cancer, it has been shown to promote tumor-mesothelial cell interactions and peritoneal metastasis by stimulating IL-1β-mediated NF-κB activity and inducing CD44 expression .

How does DLX4 influence the tumor microenvironment?

Recent research has revealed that DLX4 expression is associated with the tumor immunosuppressive microenvironment . The relationship between DLX4 and the tumor microenvironment can be assessed through:

• Immune cell infiltration: Single-sample Gene Set Enrichment Analysis (ssGSEA) using metagenes representative of 28 specific immune cell subpopulations can quantify the association between DLX4 expression and immune cell composition .

• Stromal and immune scores: The ESTIMATE package allows calculation of stromal and immune scores that reflect the infiltration levels of non-tumor components in the tumor microenvironment .

• DLX4 as an immune regulation mediator: Research suggests that DLX4 may function as a master regulator of immune infiltration recruitment, with loss of DLX4 expression potentially contributing to immune evasion in cancer .

What are the optimal protocols for immunohistochemical detection of DLX4?

For reliable immunohistochemical detection of DLX4 in paraffin-embedded tissues, the following protocol is recommended:

• Tissue preparation:

  • Cut paraffin-embedded tissue specimens into 4-μm-thick sections

  • Bake slides at 60°C for 20 minutes for dewaxing

  • Hydrate sections through xylene and graded alcohols (100%, 95%, 90%, 80%, 70%)

• Antigen retrieval:

  • Heat slides at 100°C for 10 minutes in citric acid buffer (0.01 M, pH 6.0)

• Blocking and antibody incubation:

  • Incubate slides in 3% hydrogen peroxide solution for 15 minutes at room temperature

  • Wash three times with PBS (pH 7.4)

  • Block with 3% bovine serum albumin for 1 hour at room temperature

  • Incubate with anti-DLX4 antibody (recommended dilutions: 1:50-1:200) overnight at 4°C

• Detection and visualization:

  • Incubate with biotinylated secondary antibody (e.g., goat anti-rabbit IgG at 1:200) for 2 hours at room temperature

  • Detect immune complexes using DAB

  • Counterstain with hematoxylin for 3 minutes at room temperature

• Analysis:

  • Capture images using a light microscope

  • Measure DLX4 expression based on the intensity of immune staining using image analysis software such as ImageJ

How should researchers optimize Western blot protocols for DLX4 detection?

For optimal Western blot detection of DLX4:

• Sample preparation:

  • Extract protein from tissues (e.g., human heart tissue, placenta) or cell lines (e.g., SW480, K562, HeLa)

  • Standardize protein loading (10 μg recommended)

• Antibody dilution:

  • For polyclonal antibodies: 1:200-1:2000 dilution range is recommended

  • For monoclonal antibodies: Follow manufacturer's recommendation (typically 1:1000)

• Molecular weight considerations:

  • The predicted molecular weight for DLX4 is 26 kDa

  • Observed molecular weight may vary (e.g., 38 kDa has been reported)

  • These differences may reflect post-translational modifications or alternative splice variants

• Positive controls:

  • Human heart tissue, SW480 cells, K562 cells, and placenta tissue have shown positive results

What methods are available for quantifying DLX4 gene expression?

For quantitative analysis of DLX4 gene expression:

• Quantitative PCR (qPCR):

  • Extract RNA and synthesize cDNA following standard protocols

  • Perform qPCR using SYBR Green Mix

  • Recommended thermocycling conditions: initial denaturation at 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec, with final extension at 95°C for 15 sec, 60°C for 1 min, and 95°C for 15 sec

  • Use the 2^-ΔΔCq method for relative expression analysis, with GAPDH as endogenous control

• Primer sequences:

  • DLX4 forward: 5′-CAGCACCTAAACCAGCGTTTC-3′

  • DLX4 reverse: 5′-GAGCTTCTTATACTTGGAGCGTT-3′

  • GAPDH forward: 5′-GGGAGCCAAAAGGGTCAT-3′

  • GAPDH reverse: 5′-GAGTCCTTCCACGATACCAA-3′

How can researchers address inconsistent DLX4 antibody staining?

When confronting inconsistent DLX4 antibody staining, consider these troubleshooting approaches:

• Antibody validation:

  • Verify antibody specificity using positive controls (human lung cancer and thyroid cancer tissues have shown reliable positivity)

  • Include appropriate negative controls and blocking peptides to confirm specificity

• Antigen retrieval optimization:

  • Test different antigen retrieval methods (heat-induced vs. enzymatic)

  • Optimize buffer conditions and retrieval duration

• Antibody dilution titration:

  • Perform dilution series to determine optimal concentration

  • Polyclonal antibodies typically work at 1:20-1:200 for IHC applications

• Sample fixation consideration:

  • Overfixation or insufficient fixation can affect epitope accessibility

  • Standardize fixation protocols across samples to minimize variability

• Cell/tissue type specificity:

  • Be aware that DLX4 expression varies across different cell types

  • DLX4 typically shows nuclear localization, so assess subcellular staining patterns carefully

How should researchers interpret DLX4 expression in relation to clinical parameters?

When analyzing DLX4 expression in clinical samples:

How can discrepancies between different DLX4 detection methods be reconciled?

When confronted with discrepancies between different detection methods:

What are emerging applications of DLX4 in precision medicine?

DLX4's potential as a prognostic and diagnostic biomarker in ccRCC suggests several promising directions for precision medicine:

• Therapeutic target development:

  • Investigate whether targeting DLX4 or its downstream pathways could provide therapeutic benefits

  • Explore the relationship between DLX4 expression and response to existing therapies

• Patient stratification:

  • Develop standardized DLX4 expression assays for clinical application

  • Validate cutoff values for stratifying patients into prognostic groups

• Liquid biopsy development:

  • Explore whether circulating DLX4 protein or DLX4-expressing cells could serve as minimally invasive biomarkers

  • Correlate circulating markers with tissue expression and clinical outcomes

How can DLX4 research contribute to understanding tumor immunology?

DLX4's association with the tumor immunosuppressive microenvironment suggests several research directions:

• Immune cell recruitment mechanisms:

  • Further investigate DLX4's role as a master regulator of immune infiltration recruitment

  • Explore how loss of DLX4 expression may contribute to immune evasion in cancer

• Inflammatory signaling pathways:

  • Study DLX4's role in IL-1β and NF-κB signaling in different tumor types

  • Investigate how these pathways contribute to tumor progression and metastasis

• Immunotherapy response prediction:

  • Determine if DLX4 expression levels correlate with response to immunotherapies

  • Explore combination approaches targeting DLX4 alongside immune checkpoint inhibitors