DAB2IP Antibody

What is DAB2IP and what are its main functions in cellular processes?

DAB2IP is a member of the RasGTPase-activating protein family that functions as a scaffold protein implicated in various signaling pathways. It plays crucial roles in:

• Tumor suppression by preventing cell proliferation and epithelial-to-mesenchymal transition (EMT)

• Inhibition of PI3K-AKT and Ras-MAPK survival signaling cascades

• Regulation of innate immune response and inflammatory processes

• Control of cell cycle checkpoints (reducing G1 phase cyclin levels resulting in G0/G1 arrest)

• Modulation of apoptotic pathways via ASK1-JNK signaling

• Regulation of angiogenesis and cell migration

DAB2IP achieves these functions through its multiple domains, including pleckstrin homology (PH), PKC-conserved region 2 (C2), Ras-GTPase activating protein (GAP) domain, C-terminal period-like (PER) domain, and proline-rich region that interact with different signaling molecules .

How do researchers characterize DAB2IP expression patterns in normal versus cancer tissues?

DAB2IP expression varies significantly between normal tissues and cancer tissues:

• Normal tissues: High expression in colon (RPKM 8.5), testis (RPKM 8.5), kidney (RPKM 7.1), endothelial cells, vascular smooth muscle cells (VSMCs), and prostate epithelial cells

• Cancer tissues: Expression is frequently reduced or lost in multiple cancer types including prostate, breast, ovarian, gastrointestinal, and lung cancers

Interestingly, DAB2IP shows differential expression across cancer subtypes. For example, in renal cell carcinoma (RCC):

• Higher expression in kidney chromophobe (KICH) compared to normal tissues

• Lower expression in kidney renal clear cell carcinoma (KIRC) and kidney renal papillary cell carcinoma (KIRP)

For accurate characterization, researchers typically employ multiple methods including:

• RT-qPCR for mRNA quantification

• Western blot for protein expression analysis

• Immunohistochemistry for tissue localization

• Public database analysis (e.g., UALCAN, TIMER) for larger-scale expression profiling

What are the standard applications of DAB2IP antibodies in cancer research?

DAB2IP antibodies are versatile tools employed in multiple research applications:

DAB2IP antibodies have been successfully used to detect expression in numerous cancer and normal cell lines including A375, B16, Hela, H460, H1299, MRC-5, HBE, and HUVEC, as well as tissues such as cervical cancer, bone tumor, pulmonary adenocarcinoma, and spleen .

How should researchers design experiments to study DAB2IP's role in cancer progression?

Designing experiments to elucidate DAB2IP's functions requires multi-dimensional approaches:

Genetic manipulation strategies:

• Knockdown models: Use lentivirus vector-based shRNA to create stable DAB2IP-depleted cell lines (e.g., SCL-1 in cutaneous squamous cell carcinoma studies)

• Overexpression models: Transfect DAB2IP expression vectors to restore function in cancer cells with low endogenous expression (e.g., Bxpc3-DAB2IP pancreatic cancer cells)

• Mouse models: DAB2IP knockout mice (DAB2IP-/-) develop prostate hyperplasia by 6 months of age with hyperactivated Akt and suppressed ASK1, mimicking effects seen in human cancers

Functional assays:

• Cell proliferation: MTT assays, colony formation tests

• Cell cycle analysis: Flow cytometry

• Apoptosis assessment: TUNEL staining

• Migration and invasion: Transwell assays, wound-healing assays

• In vivo tumor growth: Subcutaneous xenograft models

Signaling pathway analysis:

• Investigate Ras-MAPK pathway using Ras activity assays

• Examine PI3K-AKT survival signaling via phosphorylation status

• Study ASK1-JNK apoptotic signaling and NF-κB pathways

• Evaluate connections to HIF-1α and glucose metabolism

Comprehensive experimental designs should include appropriate controls and multiple cell lines to account for context-dependent functions of DAB2IP.

What considerations are crucial when selecting and validating DAB2IP antibodies?

Selecting appropriate DAB2IP antibodies requires careful consideration:

Antibody validation criteria:

• Specificity: Confirm using Western blot by:

  • Comparing DAB2IP-expressing vs. knockout/knockdown samples

  • Checking for correct molecular weight (118-132 kDa)

  • Pre-absorption tests with immunizing peptide

• Epitope selection: Different antibodies recognize distinct regions:

  • N-terminal epitopes (PH domain)

  • Central regions (GAP domain)

  • C-terminal epitopes (850-900 aa region in human DAB2IP)

• Species reactivity: Verify cross-reactivity with target species:

  • Human and mouse reactivity is common

  • Some antibodies offer broader reactivity including rat, pig, bovine

• Application suitability: Not all antibodies work equally well across applications:

  • Some are optimized for Western blot but perform poorly in IHC

  • Others may be specifically developed for immunoprecipitation

• Quantitative validation: Measure antibody affinity constant:

  • For example, a high-affinity anti-DAB2IP MAb had affinity constant of 2.8×10^8 L/mol

Investigators should conduct preliminary testing with positive controls known to express DAB2IP (e.g., brain tissue for Western blot, vascular smooth muscle cells for IHC) .

How can DAB2IP antibodies be utilized to investigate tumor immune microenvironment?

DAB2IP plays important roles in regulating immune responses, making antibodies valuable tools for tumor microenvironment studies:

Approaches for immune microenvironment analysis:

• Multiplex immunohistochemistry:

  • Co-stain for DAB2IP and immune cell markers (B cells, T cells, macrophages)

  • Evaluate spatial relationships between DAB2IP-expressing cells and immune infiltrates

• Flow cytometry:

  • Use DAB2IP antibodies in combination with immune cell surface markers

  • Assess correlations between DAB2IP expression and immune cell populations

• Database correlations:

  • Analyze TIMER platform data showing relationships between DAB2IP expression and tumor immune infiltration

  • For example, positive correlations between DAB2IP expression and tumor purity in KICH, KIRC, and KIRP samples have been documented

Research findings:

• DAB2IP loss in KRAS-mutant colorectal cancers triggers production of inflammatory mediators and recruitment of protumorigenic macrophages

• Infiltrating T cells may promote RCC cell invasion via decreased DAB2IP expression

• DAB2IP loss is associated with enrichment of macrophage and inflammatory signatures in human tumors

Researchers should integrate DAB2IP analysis with immune profiling to understand its role in shaping the tumor immune microenvironment.

What methodologies are most effective for developing new monoclonal antibodies against DAB2IP?

Development of novel monoclonal antibodies against DAB2IP can follow established hybridoma techniques:

Step-by-step methodology:

• Immunogen selection and preparation:

  • Synthesize human DAB2IP polypeptide (successful example: disabled homolog 2-interacting protein isoform 3)

  • Prepare with adjuvant for immunization

• Animal immunization:

  • Immunize BALB/c mice with synthesized peptide

  • Use multiple booster injections to enhance immune response

• Hybridoma generation:

  • Isolate spleen cells from immunized mice

  • Fuse with myeloma cells using polyethylene glycol

  • Culture in HAT medium for hybrid cell selection

• Screening and selection:

  • Use ELISA with synthesized DAB2IP polypeptide as coating antigen (1 μg/mL)

  • Test hybridoma supernatants as primary antibody

  • Detect with HRP-conjugated secondary antibody and TMB substrate

  • Measure optical density at 450nm to identify positive clones

• Subcloning and expansion:

  • Subclone positive hybridomas by limiting dilution

  • Expand selected clones for antibody production

• Ascites production and purification:

  • Inject hybridoma cells into peritoneal cavity of mice

  • Collect ascites after 10-14 days

  • Purify using ammonium sulfate precipitation and protein-G affinity chromatography

• Validation:

  • Determine antibody isotype and specificity

  • Measure affinity constant by non-competitive enzyme immunoassay

  • Confirm reactivity across multiple applications (Western blot, IHC, ICC)

This approach has successfully yielded antibodies with high specificity and affinity for human DAB2IP .

How do researchers interpret contradictory findings regarding DAB2IP expression and function in different cancer types?

Interpreting contradictory findings regarding DAB2IP requires careful analysis:

Sources of contradictions:

• Tissue-specific expression patterns:

  • DAB2IP shows variable baseline expression across tissues

  • In renal cell carcinoma, DAB2IP is upregulated in KICH but downregulated in KIRC and KIRP compared to normal tissues

• Context-dependent functions:

  • DAB2IP's role may differ based on genetic background of cancer cells

  • In KRAS-mutant colorectal cancers, DAB2IP functions as a bifunctional tumor suppressor that regulates both RAS signaling and inflammatory cascades

• Technical variability:

  • Different antibodies target distinct epitopes, potentially detecting different isoforms

  • Various detection methods have different sensitivity thresholds

  • Sample preparation techniques may affect antigen integrity

• Methodological approaches:

  • Studies examining mRNA versus protein levels may yield different results due to post-transcriptional regulation

  • In situ analysis versus homogenized sample analysis may show different patterns

Reconciliation strategies:

• Employ multiple antibodies targeting different epitopes

• Combine mRNA and protein expression analysis

• Use both in vitro and in vivo models

• Validate findings across multiple cancer cell lines

• Consider the role of tumor microenvironment in modulating DAB2IP functions

• Account for genetic background variations between cancer models

For example, the apparently contradictory finding that DAB2IP expression is higher in cutaneous squamous cell carcinoma (cSCC) than in soft fibroma while being reduced in many other cancers highlights the complexity of DAB2IP regulation and the need for comprehensive analytical approaches.

How can DAB2IP antibodies be optimized for diagnostic and prognostic applications in pathology?

Optimizing DAB2IP antibodies for clinical pathology requires:

Protocol standardization:

• Tissue processing:

  • Standardize fixation methods (e.g., 10% formalin for 24-48 hours)

  • Control antigen retrieval (successful protocol: 0.25% pancreatin at 37°C for 15 min, or heating at 95°C for 30 min)

• Staining protocol:

  • Establish optimal antibody dilutions (e.g., 1:100 for IHC)

  • Standardize incubation conditions (e.g., 4°C overnight)

  • Select appropriate detection systems (e.g., ABC systems, DAB stain)

• Scoring systems:

  • Implement consistent evaluation criteria

  • Example scoring system:

    • Percent positivity: 0-5%, 6-25%, 25-50%, 51-75%, or 75-100%

    • Staining intensity: no staining (0), pale yellow (1), brown yellow (2), dark yellow/tan (3)

    • Total score calculation: sum of percent positivity and intensity scores

    • Final evaluation: negative (-), weakly positive (+), moderately positive (++), strongly positive (+++)

Clinical validation:

• Correlate DAB2IP expression with patient outcomes across large cohorts

• Assess relationship with existing prognostic markers

• Perform multivariate analysis to establish independent prognostic value

• Example: Five-year survival rates significantly improved with high expression of DAB2IP in urothelial carcinoma of the bladder after surgery

Researchers should aim to establish tissue-specific thresholds for DAB2IP positivity that correlate with clinical outcomes.

What are the most promising research directions combining DAB2IP antibodies with therapeutic approaches?

DAB2IP antibodies can facilitate several therapeutic research directions:

Patient stratification for targeted therapies:

• Using DAB2IP expression to identify patients likely to respond to specific treatments

• Example: Overexpression of DAB2IP enhanced sensitivity of pancreatic cancer cells to cetuximab, suggesting DAB2IP status could predict response

Therapeutic target identification:

• Studying DAB2IP interactions to identify druggable nodes in its signaling network

• Investigating synthetic lethality opportunities when DAB2IP is lost

Immune therapy connections:

• Exploring DAB2IP's role in modulating tumor immune microenvironment

• Example: In colorectal cancer, tumor growth was suppressed by depleting macrophages or inhibiting cytokine/inflammatory mediator expression with JAK/TBK1 inhibitor in DAB2IP-deficient tumors

Metabolism-directed approaches:

• Targeting metabolic vulnerabilities created by DAB2IP loss

• Example: DAB2IP inhibits glucose uptake under hypoxia by suppressing HIF-1α signaling in breast cancer, suggesting glucose metabolism inhibitors may be effective in DAB2IP-deficient tumors

Resistance mechanisms:

• Understanding how DAB2IP status affects treatment resistance

• Example: Low DAB2IP expression contributes to mTOR-targeted therapy resistance in renal cell carcinoma

These research directions can guide the development of precision medicine approaches based on DAB2IP status in different cancers.

What are common technical challenges when using DAB2IP antibodies and how can they be addressed?

Researchers frequently encounter several technical issues when working with DAB2IP antibodies:

When troubleshooting, it's crucial to include appropriate controls:

• Positive controls: Brain tissue for Western blot , vascular smooth muscle cells for IHC

• Negative controls: DAB2IP knockdown samples, isotype controls, omitting primary antibody

How can researchers effectively compare and interpret DAB2IP expression data from different antibody-based techniques?

Comparing data from different antibody-based techniques requires methodological considerations:

Cross-technique standardization:

• Use common reference samples:

  • Include the same positive and negative controls across all techniques

  • Analyze a gradient of DAB2IP expression levels to establish technique-specific sensitivity

• Account for technique-specific limitations:

  • Western blot: Quantitative but loses spatial information

  • IHC/ICC: Preserves spatial context but semi-quantitative

  • ELISA: Highly quantitative but lacks cellular context

  • IP: Detects native protein interactions but not always quantitative

• Data normalization strategies:

  • Western blot: Normalize to loading controls (β-actin, GAPDH)

  • IHC: Use standardized scoring systems like H-score or Allred score

  • Compare relative rather than absolute expression levels between techniques

• Concordance assessment:

  • Calculate correlation coefficients between techniques

  • Perform Bland-Altman analysis to identify systematic biases

  • Use statistical methods appropriate for categorical vs. continuous data

When interpreting discrepancies, consider that:

• Different techniques may detect different isoforms or phosphorylation states

• Antibodies might have different specificities and affinities

• Sample preparation affects epitope accessibility

• Detection thresholds vary between methods

How might emerging technologies enhance the utility of DAB2IP antibodies in cancer research?

Emerging technologies offer new opportunities for DAB2IP antibody applications:

Single-cell analysis:

• Single-cell Western blotting for heterogeneity assessment

• Mass cytometry (CyTOF) with metal-conjugated DAB2IP antibodies

• Single-cell spatial transcriptomics combined with protein detection

Advanced imaging:

• Super-resolution microscopy to visualize DAB2IP interactions at nanoscale

• Intravital microscopy to track DAB2IP dynamics in living tissues

• Proximity ligation assays to detect protein-protein interactions in situ

Antibody engineering:

• Bispecific antibodies targeting DAB2IP and pathway components

• Recombinant antibody fragments with enhanced tissue penetration

• Site-specific conjugation for precisely controlled reporter attachment

High-throughput screening:

• Antibody arrays for rapid profiling of DAB2IP and related proteins

• Microfluidic platforms for automated antibody validation

• AI-assisted image analysis for quantitative evaluation of staining patterns

These technologies could provide unprecedented insights into DAB2IP's dynamic regulation and context-dependent functions in cancer progression.

What research gaps remain in understanding DAB2IP's roles that could be addressed with improved antibody tools?

Despite significant progress, several research gaps could be addressed with improved antibody tools:

Isoform-specific functions:

• Development of isoform-specific antibodies to distinguish between DAB2IP variants

• Investigation of tissue-specific expression patterns of different isoforms

• Elucidation of isoform-specific protein interactions and signaling outcomes

Post-translational modifications:

• Generation of antibodies specific to phosphorylated, ubiquitinated, or otherwise modified DAB2IP

• Mapping of modification-dependent protein interactions

• Temporal dynamics of DAB2IP modifications during cancer progression

Context-dependent mechanisms:

• Tools to study DAB2IP in the context of cell density and mechanical inputs

• Antibodies optimized for detecting DAB2IP in different subcellular compartments

• Methods to visualize DAB2IP conformational changes upon activation

Tumor microenvironment interactions:

• Multiplex approaches to simultaneously detect DAB2IP and immune/stromal markers

• Investigation of DAB2IP's role in intercellular communication

• Assessment of DAB2IP status in circulating tumor cells and extracellular vesicles

Addressing these gaps would provide a more comprehensive understanding of DAB2IP's complex roles in normal physiology and cancer pathogenesis, potentially revealing new therapeutic opportunities.