DAB2 Antibody

What is DAB2 and what biological functions does it serve?

DAB2 (Disabled homolog 2) is a multifunctional adapter protein involved in several critical cellular processes including receptor-mediated signaling, endocytosis, cell adhesion, hematopoietic cell differentiation, and angiogenesis . At the molecular level, DAB2 functions as an endocytic adaptor protein that mediates clathrin-dependent endocytosis of various cell surface receptors. The protein plays significant roles in TGF-β signaling pathways, where it regulates the domain localization of TGF-β type I receptor (TβRI) in the plasma membrane, thereby balancing signaling through Smad and JNK pathways . Additionally, DAB2 has emerged as an important regulator of dendritic cell (DC) function, where it appears to act as a negative regulator of DC immunogenicity .

What are the available forms of DAB2 antibodies for research applications?

Research-grade DAB2 antibodies are available in various formats optimized for different experimental applications. For instance, monoclonal antibodies such as the Mouse Anti-Human DAB2 Monoclonal Antibody (Clone # 883216) are derived from E. coli-expressed recombinant human DAB2 (specifically amino acids Lys630-Ala770) . These antibodies have been validated for applications including Western blot, immunocytochemistry (ICC), and immunohistochemistry (IHC) . When selecting a DAB2 antibody, researchers should consider the specific epitope recognized, species reactivity, clonality (monoclonal vs. polyclonal), and whether the antibody has been validated for their particular application and experimental system.

How is DAB2 expressed in different cell types and tissues?

DAB2 exhibits a diverse expression pattern across cell types and tissues. In the immune system, DAB2 is significantly expressed during GM-CSF-mediated bone marrow-derived dendritic cell (BMDC) development . Expression increases in parallel with CD11c (a dendritic cell marker) during BMDC differentiation . DAB2 is also highly expressed in primary MHC II high CD11c+ splenic dendritic cells isolated from normal mice and in human monocyte-derived dendritic cells (MoDCs) . Beyond immune cells, DAB2 expression has been detected in various cell lines including HeLa human cervical epithelial carcinoma cells and A172 human glioblastoma cells . In tissues, DAB2 has been localized to the cytoplasm of epithelial cells in human prostate tissue . The expression pattern suggests tissue-specific and context-dependent roles for DAB2 in cellular function.

What are the validated applications for DAB2 antibodies in cellular imaging?

DAB2 antibodies have been successfully employed for various cellular imaging applications, providing valuable insights into DAB2 localization and function. For immunocytochemistry (ICC), researchers have used anti-DAB2 monoclonal antibodies (at concentrations of approximately 10 μg/mL) with fixed cells, such as HeLa human cervical epithelial carcinoma cells, resulting in specific cytoplasmic staining . The protocol typically involves cell fixation, permeabilization, blocking, overnight primary antibody incubation, and visualization using fluorophore-conjugated secondary antibodies (such as NorthernLights™ 557-conjugated Anti-Mouse IgG) . Nuclear counterstaining with DAPI helps contextualize DAB2 localization within cells. For optimal results, researchers should optimize antibody concentration, incubation time, and temperature for their specific cell type and fixation method.

How should DAB2 antibodies be used for Western blot analysis?

For Western blot applications, DAB2 antibodies can detect the protein at approximately 100 kDa under reducing conditions . A validated protocol involves preparing cell lysates (from sources such as A172 human glioblastoma cells or HeLa cells), separating proteins via SDS-PAGE, transferring to PVDF membranes, and probing with anti-DAB2 antibodies at 1 μg/mL concentration . Detection is typically achieved using HRP-conjugated secondary antibodies followed by chemiluminescent substrate. Critical considerations for successful Western blot include using appropriate lysis buffers to solubilize DAB2, optimizing blocking conditions to minimize background, and selecting suitable positive controls. For quantitative analysis, researchers should include loading controls and consider the dynamic range of detection when interpreting band intensities.

What methodology is recommended for DAB2 detection in tissue samples?

For detecting DAB2 in tissue sections, immunohistochemistry (IHC) using paraffin-embedded sections has proven effective. A validated approach includes tissue fixation, embedding, sectioning, deparaffinization, antigen retrieval, and overnight incubation with anti-DAB2 antibodies at 15 μg/mL at 4°C . Detection systems such as HRP-DAB (3,3'-diaminobenzidine) produce a brown precipitate at sites of DAB2 expression, with hematoxylin counterstaining providing structural context . When examining epithelial tissues such as prostate, DAB2 typically localizes to the cytoplasm of epithelial cells . Researchers should include appropriate positive and negative controls and may need to optimize antigen retrieval methods (heat-induced vs. enzymatic) depending on the specific tissue and fixation protocol. Quantitative assessment of DAB2 expression in tissues may be achieved through digital image analysis using appropriate software.

How does DAB2 influence dendritic cell function and immunogenicity?

DAB2 appears to function as an intrinsic negative regulator of dendritic cell (DC) immunogenicity. Experimental silencing of DAB2 in DCs leads to enhanced expression of MHC I, MHC II, and co-stimulatory molecules CD40 and CD80 in both immature and mature DCs . This phenotypic modification correlates with functional changes, as DAB2-silenced DCs demonstrate increased antigen uptake capacity and enhanced migration capability . At the molecular level, DAB2 knockdown upregulates Th1 cytokines like IL-12 and IL-6, which significantly improves the DCs' T cell stimulation capacity, leading to stronger cytotoxic T lymphocyte (CTL) responses in vaccinated mice . Consequently, vaccination with DAB2-silenced DCs inhibits tumor growth more effectively than vaccination with wild-type DCs, while DAB2 overexpression abrogates DC vaccine efficacy . These findings establish DAB2 as a potential molecular target for improving DC-based immunotherapies.

What is the relationship between DAB2 and TGF-β signaling pathways?

DAB2 plays a critical regulatory role in TGF-β signaling pathways by modulating receptor localization and downstream pathway activation. Mechanistically, DAB2 interacts directly with the type I TGF-β receptor (TβRI) to restrict its lateral diffusion at the plasma membrane and enhance its clathrin-mediated endocytosis . This spatial regulation selectively impacts TGF-β signaling outputs: while DAB2 negatively regulates TGF-β-induced c-Jun N-terminal kinase (JNK) activation, it does not affect activation of the canonical Smad pathway . The differential regulation appears to be cholesterol-dependent, as disruption of membrane cholesterol eliminates JNK activation by TGF-β in the absence of DAB2 . These findings support a model in which DAB2 functions as a molecular switch that balances TGF-β signaling through different downstream pathways by controlling the membrane domain localization of TGF-β receptors, with significant implications for understanding cellular responses to TGF-β in various physiological and pathological contexts.

How is DAB2 expression regulated by Toll-like receptor activation?

Toll-like receptor (TLR) activation induces rapid downregulation of DAB2 in dendritic cells through both transcriptional and post-translational mechanisms. Exposure to TLR ligands, including those that activate TLR1-9 (with the exception of TLR5), significantly reduces DAB2 protein expression in bone marrow-derived dendritic cells (BMDCs) . This downregulation involves both major TLR adapter proteins—MyD88 and TRIF—as demonstrated by the abolishment of DAB2 downregulation in BMDCs from MyD88⁻/⁻ mice treated with TLR2 agonists and in BMDCs from TRIF⁻/⁻ mice treated with TLR3 agonists . Kinetic analyses reveal that LPS exposure triggers a rapid decline in DAB2 protein within 60 minutes, preceding any significant changes in DAB2 mRNA levels, which gradually decrease over the subsequent 16 hours . This biphasic regulation suggests that initial DAB2 protein destabilization is followed by transcriptional repression. The physiological consequence of TLR-induced DAB2 downregulation is a more functional and activated DC phenotype, characterized by reduced phagocytosis and increased CD40 expression .

What approaches can be used to manipulate DAB2 expression for functional studies?

Several effective strategies exist for modulating DAB2 expression to investigate its functional roles. RNA interference using DAB2-specific siRNA has proven successful in reducing DAB2 expression by over 80% at both mRNA and protein levels in dendritic cells . This approach allows for transient knockdown suitable for acute functional studies. For studies requiring stable DAB2 suppression, shRNA-expressing lentiviral vectors can be employed. Conversely, stable overexpression of DAB2 has been achieved using appropriate expression vectors, as demonstrated in the ES-2 cell line . CRISPR-Cas9 gene editing presents an alternative for complete DAB2 knockout or for introducing specific mutations to study structure-function relationships. When investigating DAB2's role in particular signaling pathways, researchers should consider using pathway-specific inhibitors in conjunction with DAB2 manipulation. For instance, studies examining DAB2's involvement in TGF-β signaling have employed pharmacological inhibitors of JNK, PI3K, and other signaling components to delineate the specific pathways affected by DAB2 expression changes .

How should researchers design experiments to study DAB2's role in membrane dynamics?

Investigating DAB2's impact on membrane dynamics requires specialized biophysical approaches. Fluorescence recovery after photobleaching (FRAP) has been successfully employed to analyze DAB2's effect on the lateral diffusion of interacting proteins like TβRI . In this approach, fluorescently labeled receptors are transiently expressed, a small area of the membrane is photobleached, and the recovery of fluorescence is monitored over time. The diffusion coefficient (D) and mobile fraction (R) provide quantitative measures of lateral mobility and interaction dynamics . Complementary techniques include single-particle tracking to monitor the movement of individual receptor molecules and fluorescence correlation spectroscopy for diffusion measurements. To investigate DAB2's role in endocytosis, researchers can employ the point-confocal endocytosis assay, which allows quantification of internalization rates . When designing these experiments, it is crucial to include appropriate controls such as cholesterol depletion (using methyl-β-cyclodextrin) to assess membrane domain contributions, cytoskeletal disruption agents to evaluate the role of the cytoskeleton, and comparison with known endocytic adaptor proteins as functional references.

What considerations are important when interpreting phenotypic changes following DAB2 manipulation?

When analyzing phenotypic changes resulting from DAB2 manipulation, researchers should implement several experimental controls and interpretative frameworks. First, it is essential to confirm the specificity of DAB2 modulation by using multiple siRNA/shRNA sequences or rescue experiments with siRNA-resistant DAB2 constructs. Second, researchers should distinguish direct from indirect effects by examining the kinetics of phenotypic changes—rapid alterations may indicate direct DAB2 involvement, while delayed responses might suggest secondary effects. Third, since DAB2 functions in multiple cellular processes, phenotypic changes should be interpreted in the context of potential alterations in endocytosis, signaling pathway activation, and membrane dynamics. For instance, when studying DAB2's role in dendritic cells, researchers have examined multiple functional parameters including surface marker expression, cytokine production, antigen uptake, migration capacity, and T cell stimulation . Finally, researchers should consider cell type specificity, as DAB2 function may vary across different cellular contexts—findings in one cell type (e.g., dendritic cells) may not directly translate to others (e.g., epithelial cells).

How can researchers validate DAB2 antibody specificity for their experimental system?

Validating DAB2 antibody specificity is critical for generating reliable experimental results. A comprehensive validation approach should include multiple complementary strategies. First, researchers should perform Western blot analysis to confirm that the antibody detects a protein of the expected molecular weight (approximately 100 kDa for DAB2) . Second, siRNA knockdown or CRISPR knockout of DAB2 should result in corresponding reduction or elimination of the detected signal across all applications (Western blot, ICC, IHC). Third, immunoprecipitation followed by mass spectrometry can confirm that the antibody is pulling down DAB2 rather than cross-reacting proteins. Fourth, comparison of staining patterns across multiple antibodies targeting different DAB2 epitopes can provide additional confidence in specificity. Fifth, parallel analysis of DAB2 mRNA and protein expression across tissues or experimental conditions should show concordant patterns. Finally, researchers should carefully evaluate vendor validation data, including specific validation in their cell type or tissue of interest, and consider published literature utilizing the same antibody for similar applications.

What are the common technical challenges when using DAB2 antibodies for Western blot?

Researchers frequently encounter several challenges when detecting DAB2 by Western blot. First, inadequate protein extraction may occur since DAB2 functions as an adapter protein with membrane associations, potentially requiring specialized lysis buffers containing appropriate detergents to ensure complete solubilization. Second, the relatively high molecular weight of DAB2 (approximately 100 kDa) necessitates careful optimization of gel percentage and transfer conditions to ensure efficient protein transfer to membranes. Third, non-specific bands may appear, particularly in complex samples, requiring careful antibody titration and extensive blocking optimization. Fourth, when studying DAB2 in stimulation experiments (e.g., TLR activation), the rapid downregulation of DAB2 protein (occurring within 60 minutes of stimulation) demands precise timing of sample collection. Fifth, since DAB2 expression varies across cell types and differentiation states, researchers should include positive control samples with known DAB2 expression. Finally, for quantitative Western blot analysis, researchers should carefully select loading controls that remain stable under the experimental conditions, as common housekeeping proteins may be affected by treatments that alter DAB2 expression.

What controls should be included when studying DAB2 regulation by TLR or TGF-β pathways?

When investigating DAB2 regulation by TLR or TGF-β pathways, several essential controls should be incorporated. For TLR studies, researchers should include: (1) time course experiments to distinguish between early post-translational and later transcriptional effects on DAB2 expression ; (2) pathway-specific controls using cells from MyD88⁻/⁻ or TRIF⁻/⁻ mice to delineate adapter protein requirements ; (3) protein synthesis inhibitors like cycloheximide to assess DAB2 protein stability; and (4) transcription inhibitors like actinomycin D to evaluate mRNA stability . For TGF-β pathway studies, critical controls include: (1) Smad phosphorylation analysis to confirm canonical pathway activation; (2) cholesterol depletion experiments to assess membrane domain contributions to DAB2 function ; (3) comparison of DAB2 effects on multiple TGF-β-induced pathways (e.g., Smad vs. JNK) ; and (4) FRAP analysis with mutated forms of DAB2 lacking specific binding domains to identify critical protein-protein interactions. For both pathways, researchers should carefully consider the timing of analyses, as DAB2 regulation may involve biphasic responses with distinct molecular mechanisms operating at different time points.