DAPB2 Antibody

What is DAB2 protein and why is it significant in cellular research?

DAB2 protein, also known as DOC-2, is a mitogen-responsive phosphoprotein that plays crucial roles in cellular signaling pathways regulating growth and development. It primarily localizes in the cytoplasm where it functions as a negative regulator of growth, particularly in response to mitogenic signals. DAB2's significance lies in its ability to modulate growth signals, which is vital for maintaining cellular homeostasis and preventing uncontrolled cell proliferation that can lead to tumorigenesis. Its interactions with the SH3 domain of GRB2 and other signaling proteins underscore its importance in various cellular processes, including differentiation and migration. These characteristics make DAB2 protein a valuable target for research in cancer biology and developmental studies .

What detection methods can be reliably used with DAB2 antibodies?

DAB2 antibodies, such as the mouse monoclonal IgG1 kappa light chain antibody (E-11), can be utilized across multiple detection platforms. Researchers can reliably employ these antibodies for western blotting (WB), immunoprecipitation (IP), immunofluorescence (IF), immunohistochemistry with paraffin-embedded sections (IHCP), and enzyme-linked immunosorbent assay (ELISA). The versatility of DAB2 antibodies extends to both non-conjugated forms and various conjugated versions, including agarose, horseradish peroxidase (HRP), phycoerythrin (PE), fluorescein isothiocyanate (FITC), and multiple Alexa Fluor® conjugates . This flexibility allows researchers to select the most appropriate format based on their specific experimental design and detection system.

How should dendritic cell cultures be prepared for DAB2 expression studies?

For DAB2 expression studies in dendritic cells, bone marrow (BM) should be obtained from mouse femurs and tibias by flushing with cold complete RPMI medium. After lysing red blood cells using RBC lysis buffer, leukocytes should be incubated with 200 ng/mL FLT3L in complete RPMI 1640 medium containing 55 μM β-mercaptoethanol for 7 days at 37°C with 5% CO2. This should be followed by an additional 2-day incubation with complete RPMI 1640 containing 20 ng/mL GM-CSF and 200 ng/mL FLT3L to generate CD103+ dendritic cells. Harvest BMDCs for analysis on day 9 and confirm their phenotype by flow cytometry. This protocol generates a heterogeneous mix of DCs based on their CD103 and CD11b expression, allowing for comprehensive DAB2 expression analysis across different DC subsets .

What is the species reactivity profile of commonly used DAB2 antibodies?

Most commonly used research-grade DAB2 antibodies, particularly the E-11 clone, demonstrate cross-reactivity across multiple mammalian species. Specifically, these antibodies can detect DAB2 protein of mouse, rat, and human origin . This multi-species reactivity makes these antibodies particularly valuable for comparative studies and translational research where findings from animal models need to be validated in human samples. When selecting a DAB2 antibody for your research, it's important to verify the specific species reactivity in the context of your experimental design, as some applications may demonstrate differential sensitivity across species.

How does TLR activation affect DAB2 expression in dendritic cells?

TLR activation leads to a rapid and significant downregulation of DAB2 expression in dendritic cells through complex mechanisms. Upon TLR stimulation (with the exception of TLR5 activation), DAB2 protein levels rapidly decline, with maximum reduction occurring at approximately 60 minutes post-LPS exposure, well before any significant changes in DAB2 mRNA can be detected. This protein reduction is sustained for at least 24 hours after initial exposure. The DAB2 mRNA gradually decreases (by >60% compared to control cells) in LPS-treated BMDCs, with statistically significant reduction first observed at 2 hours and continuing to decline for at least 16 hours post-treatment .

This biphasic response involves both rapid protein degradation and sustained transcriptional suppression. The downregulation process requires both MyD88 and TRIF adaptor proteins, as demonstrated by abolished DAB2 downregulation in BMDCs from MyD88-/- mice treated with HKLM (TLR2 agonist) and in BMDCs from TRIF-/- mice treated with poly I:C HMW (TLR3 agonist). Notably, DAB2 downregulation by LPS is only partially restored in BMDCs from either TRIF-/- or MyD88-/- mice, since TLR4 utilizes both adapters .

What are the optimal protocols for intracellular DAB2 staining for flow cytometry?

For optimal intracellular DAB2 staining in flow cytometry applications, cells should first be stained with Fixable Viability Dye eFluor™ 506 (1:1,000) to exclude dead cells. Following surface marker staining, incubate cells in Fixation/Permeabilization Buffer for 30 minutes at 4°C. After permeabilization, treat cells with rabbit anti-DAB2 antibody (1:100 dilution) in permeabilization buffer for 1 hour at room temperature. Then incubate with Alexa Fluor 647-conjugated secondary antibody (F(ab')2-Goat anti-Rabbit IgG) at 1:1,000 dilution in permeabilization buffer for 30 minutes .

This protocol allows for simultaneous detection of surface markers and intracellular DAB2 protein. For analyzing different DC subsets, include appropriate markers such as CD11c, CD11b, and CD103 in your panel design. When analyzing results, first gate on viable single cells, then identify your population of interest before assessing DAB2 expression levels. This approach allows for precise quantification of DAB2 across different cell subpopulations.

How should researchers interpret stability differences between DAB2 protein and mRNA?

Researchers should interpret the stability differences between DAB2 protein and mRNA as indicative of complex post-transcriptional and post-translational regulatory mechanisms. Experimental data demonstrates that DAB2 protein in dendritic cells has notably low stability, with significant degradation occurring within 30 minutes of cycloheximide treatment (which blocks new protein synthesis). In contrast, DAB2 mRNA exhibits remarkable stability, as evidenced by its persistence during actinomycin D treatment (which inhibits new RNA synthesis) .

Intriguingly, cycloheximide treatment leads to increased DAB2 mRNA levels, suggesting that at baseline, DAB2 transcript levels are negatively regulated by short-lived proteins and/or miRNAs. This inverse relationship indicates a sophisticated regulatory system where protein turnover and mRNA stability are differentially controlled. When designing experiments to study DAB2 function, researchers should account for these different degradation kinetics by: (1) using short time points when studying protein dynamics, (2) considering that protein changes may precede mRNA changes, and (3) recognizing that interventions affecting protein synthesis may paradoxically increase mRNA levels due to relief of negative feedback mechanisms .

What functional changes occur in dendritic cells following DAB2 downregulation?

DAB2 downregulation in dendritic cells promotes a more functional and activated phenotype characterized by several key changes. First, DCs exhibit reduced phagocytic capacity following DAB2 downregulation, indicating a shift from antigen capture to antigen presentation functions. Second, there is increased surface expression of CD40, a co-stimulatory molecule critical for T cell activation and DC-T cell interactions .

These phenotypic changes suggest that DAB2 normally functions as a negative regulator of DC maturation and activation. When DAB2 is downregulated through TLR activation, DCs transition from an immature state focused on antigen capture to a mature state optimized for antigen presentation and T cell stimulation. This functional transition is essential for effective adaptive immune responses. Researchers investigating DC biology should consider DAB2 expression levels as a potential marker of DC maturation state and functional capacity in their experimental systems .

How can researchers effectively validate DAB2 antibody specificity?

To effectively validate DAB2 antibody specificity, researchers should implement a multi-faceted approach. Begin with a knockout/knockdown validation by comparing antibody reactivity in wild-type cells versus those with CRISPR-Cas9 modified DAB2 (as described in the DC2.4 cell model). This genetic approach provides the most definitive evidence of specificity . Additionally, perform peptide competition assays where the antibody is pre-incubated with purified DAB2 protein before application to samples—a specific antibody will show diminished or absent signal.

Western blotting should reveal a band of appropriate molecular weight (~96 kDa for full-length DAB2) with minimal non-specific bands. For immunohistochemistry or immunofluorescence applications, include appropriate negative controls (secondary antibody only, isotype control) and positive controls (tissues known to express DAB2, such as certain epithelial cells). Finally, cross-validate findings using multiple antibodies targeting different DAB2 epitopes and different detection methods. This comprehensive validation strategy ensures reliable, reproducible results when using DAB2 antibodies in research applications.

What are the optimal western blotting conditions for detecting DAB2 protein?

For optimal western blotting detection of DAB2 protein, researchers should use a 4-20% gradient Tris-Glycine protein gel to accommodate the protein's high molecular weight (~96 kDa). Following electrophoretic separation, transfer proteins to a polyvinylidene difluoride (PVDF) membrane. Block the membrane with 5% nonfat milk or BSA in TBS-T buffer. Probe with primary DAB2 antibody at a 1:1,000 dilution overnight at 4°C, followed by incubation with an HRP-conjugated anti-rabbit/mouse IgG secondary antibody at 1:5,000 dilution .

Develop blots using ECL Western Blotting Substrate and detect chemiluminescence using an imaging system. For accurate quantification, perform densitometric analysis and normalize DAB2 expression to an appropriate loading control such as β-actin (for BMDCs) or GAPDH (for DC2.4 cells). When troubleshooting weak signals, consider extending primary antibody incubation time, increasing antibody concentration, or using enhanced chemiluminescence reagents. For high background, increase blocking time or add 0.1% Tween-20 to washing buffers to reduce non-specific binding .

How should researchers design experiments to study DAB2 regulation by TLR signaling?

When designing experiments to study DAB2 regulation by TLR signaling, researchers should implement a comprehensive time-course approach that captures both immediate protein changes and longer-term transcriptional effects. Begin with short time points (15, 30, 60, 90 minutes) to capture rapid protein downregulation, and extend observations to longer periods (2, 4, 8, 16, 24 hours) to document transcriptional effects .

Include multiple TLR agonists to determine pathway specificity: LPS (TLR4), HKLM (TLR2), Poly I:C (TLR3), Flagellin (TLR5), and CpG ODN (TLR9). For mechanistic insights, incorporate inhibitors targeting specific degradation pathways (proteasomal inhibitors like MG132 and lysosomal inhibitors like bafilomycin A1). To dissect signaling requirements, utilize cells from knockout mice lacking key adaptors (MyD88-/- and TRIF-/-) or employ siRNA knockdown of these components in cell lines .

For comprehensive analysis, parallel samples should be processed for both protein analysis (western blotting, flow cytometry) and transcript analysis (qRT-PCR). Additionally, incorporate protein synthesis inhibitors (cycloheximide) and transcription inhibitors (actinomycin D) to assess protein and mRNA stability. This multifaceted experimental design will provide robust insights into the complex regulation of DAB2 by TLR signaling .

How does DAB2 expression vary across different dendritic cell subsets?

DAB2 expression exhibits notable variation across different dendritic cell subsets, with expression patterns correlating with specific functional characteristics. In bone marrow-derived dendritic cells (BMDCs) generated using FLT3L and GM-CSF, all DC subsets express DAB2, but CD11b+ BMDCs consistently display the highest expression levels regardless of CD103 expression status . This differential expression pattern suggests that DAB2 may play specialized regulatory roles in specific DC subpopulations.

The elevated DAB2 expression in CD11b+ DCs is particularly intriguing given that these cells typically exhibit distinct antigen presentation capabilities and cytokine production profiles compared to CD103+ DCs. Since CD11b+ DCs are often associated with MHC-II presentation and CD4+ T cell responses, while CD103+ DCs excel at cross-presentation to CD8+ T cells, the preferential expression of DAB2 in CD11b+ populations may indicate a selective regulatory role in certain antigen presentation pathways. Researchers investigating DC heterogeneity should consider DAB2 as a potential marker and functional regulator that may contribute to the specialized functions of different DC subsets .

What is the relationship between DAB2 expression and dendritic cell maturation state?

The relationship between DAB2 expression and dendritic cell maturation state appears to be inversely correlated, with DAB2 functioning as a molecular brake on DC activation. Immature DCs, which specialize in antigen capture and processing, express relatively high levels of DAB2. Upon receiving maturation signals through TLR activation, DCs rapidly downregulate DAB2 protein, followed by sustained mRNA suppression .

This downregulation coincides with acquisition of mature DC characteristics, including reduced phagocytosis (shifting from antigen capture to presentation) and increased CD40 expression (enhancing co-stimulatory capacity for T cell activation). The functional significance of this relationship is highlighted by studies showing that DAB2 downregulation promotes a more activated DC phenotype . Researchers can leverage this relationship by using DAB2 expression as a molecular indicator of DC maturation status, complementing traditional surface markers. Additionally, this inverse relationship suggests that targeted manipulation of DAB2 expression might represent a novel approach to modulate DC maturation for therapeutic applications in vaccines or immunotherapies.

What potential roles might DAB2 play in immune response regulation beyond dendritic cells?

While current research has focused on DAB2's role in dendritic cells, its function as a negative regulator of cellular activation suggests potential broader involvement in immune response regulation. DAB2's ability to modulate growth signals and prevent uncontrolled cell proliferation may extend to regulating proliferation and activation of other immune cell populations, including T cells, B cells, and macrophages. Its interaction with the SH3 domain of GRB2 places DAB2 at a critical juncture in cell signaling pathways that are universally important across immune cell types.

The rapid downregulation of DAB2 following TLR activation in DCs suggests it may serve as a molecular switch controlling the transition between resting and activated states in multiple immune cell types. Furthermore, given DAB2's role in differentiation and migration , it may influence immune cell development in bone marrow and thymus, as well as trafficking to sites of inflammation. Future research should explore DAB2 expression patterns across the immune system and investigate whether the regulatory mechanisms observed in DCs are conserved in other leukocyte populations.

What control samples should be included when using DAB2 antibodies in immunostaining experiments?

When using DAB2 antibodies in immunostaining experiments, researchers should include multiple controls to ensure specificity and reliability of results. First, incorporate a genetic negative control using DAB2 knockout or knockdown cells/tissues whenever possible, as this provides the most definitive validation of antibody specificity . Second, include an isotype control antibody matched to the DAB2 primary antibody's species and immunoglobulin class to identify any non-specific binding due to Fc receptor interactions or other non-specific binding mechanisms.

Third, include a secondary-only control (omitting primary antibody) to evaluate background from the detection system. Fourth, include positive control samples from tissues known to express DAB2 (e.g., certain epithelial cells) to confirm staining procedures are working properly. Fifth, for quantitative studies, include a titration series of primary antibody concentrations to determine optimal signal-to-noise ratio. Finally, when studying DAB2 downregulation after stimulation, always include both untreated and treated samples processed in parallel to accurately assess relative expression changes. This comprehensive control strategy ensures robust, reproducible results when using DAB2 antibodies for immunostaining applications.

How should researchers design time-course experiments to study DAB2 regulation?

When designing time-course experiments to study DAB2 regulation, researchers should adopt a biphasic approach that captures both rapid protein-level changes and longer-term transcriptional regulation. For protein dynamics, include very early time points (5, 15, 30, 60, 90 minutes post-stimulation) to capture the rapid downregulation phase, as DAB2 protein exhibits maximum reduction at approximately 60 minutes following LPS exposure . For transcriptional regulation, extend observations to longer intervals (2, 4, 8, 16, 24 hours) to document the gradual reduction in DAB2 mRNA levels, which begins to show statistically significant changes at 2 hours post-stimulation .

To comprehensively monitor both protein and transcript dynamics, collect parallel samples for western blotting and qRT-PCR at each time point. Include appropriate housekeeping controls (β-actin for protein, TBP for mRNA) for normalization . To distinguish between different regulatory mechanisms, incorporate protein synthesis inhibitors (cycloheximide) and transcription inhibitors (actinomycin D) in parallel experimental arms. This design will help determine whether observed changes result from altered synthesis, degradation, or both. Finally, synchronize cell populations before stimulation (e.g., by serum starvation) to minimize variation from cell cycle effects on DAB2 expression.

What considerations are important when using DAB2 antibodies for co-immunoprecipitation studies?

When using DAB2 antibodies for co-immunoprecipitation (co-IP) studies, several critical considerations must be addressed to ensure successful protein complex isolation. First, select an appropriate DAB2 antibody format—non-conjugated or agarose-conjugated antibodies are preferred for co-IP applications . The antibody should recognize native (non-denatured) DAB2 protein and demonstrate high specificity in immunoprecipitation validation studies.

Cell lysis conditions are crucial; use gentle non-ionic detergents (0.5-1% NP-40 or Triton X-100) to preserve protein-protein interactions while effectively solubilizing membrane-associated complexes. Include protease inhibitors, phosphatase inhibitors, and maintain cold temperature throughout to prevent complex dissociation and protein degradation. Pre-clear lysates with protein A/G beads to reduce non-specific binding before adding DAB2 antibody.

For controls, perform parallel IPs with isotype-matched control antibodies to identify non-specific interactions. Additionally, include DAB2 knockout/knockdown samples as negative controls . When analyzing co-IP results, confirm successful DAB2 precipitation before examining potential interacting partners. Finally, consider crosslinking approaches for transient interactions, and reciprocal co-IPs (immunoprecipitating with antibodies against suspected interacting partners and blotting for DAB2) to validate interactions.