DAAM2 Antibody

What is DAAM2 and what is its biological significance?

DAAM2 is a member of the formin family of actin-modulating proteins that functions as a key regulator of the Wnt signaling pathway. It acts downstream of Wnt ligands and upstream of beta-catenin (CTNNB1), promoting the aggregation of Disheveled (Dvl) complexes and formation of Wnt receptor signalosomes . DAAM2 is essential for various developmental processes including dorsal patterning, determination of left/right symmetry, and myelination in the central nervous system . Additionally, it regulates actin nucleation and elongation, filopodia formation, and podocyte migration .

The protein's significance extends beyond development to potential roles in disease contexts. Recent research has identified DAAM2 as a potential biomarker in cancer immunology, particularly in pancreatic adenocarcinoma (PAAD), where it is associated with an "immuno-hot" phenotype and may predict therapeutic responses .

In which tissues is DAAM2 expression most prominent?

DAAM2 expression has been documented in multiple tissues across species:

DAAM2 is also expressed in the developing gut and dorsal mesentery, where it plays a role in left/right asymmetry establishment . In the central nervous system, it is expressed in oligodendrocytes, where it regulates myelination processes .

Which applications have been validated for anti-DAAM2 antibodies?

Current research has validated DAAM2 antibodies for multiple experimental applications:

When selecting an antibody, researchers should verify species reactivity and application suitability based on the specific experimental needs .

What are the recommended protocols for DAAM2 immunohistochemistry?

For optimal DAAM2 detection in tissue sections, the following protocol has been validated in pancreatic adenocarcinoma studies:

• Use standard IHC procedures on formalin-fixed paraffin-embedded tissues

• Apply primary anti-DAAM2 antibody at 1:200 dilution (25206-1-AP, Proteintech)

• Visualize using DAB (3,3'-diaminobenzidine) as the chromogen

• Counterstain with hematoxylin

• Scan stained sections using digital pathology slide scanners

• For quantification, employ the immunoreactivity score (IRS) system with independent assessment by two pathologists

This approach has successfully demonstrated differential DAAM2 expression between tumor and paratumor tissues in pancreatic cancer studies .

What positive and negative controls should be used when working with DAAM2 antibodies?

Based on published research, the following controls are recommended:

Positive Controls:

• Western Blot: Mouse or rat brain tissue extracts

• Overexpression systems: Transfected HEK-293 cells expressing DAAM2

• IHC: Mouse heart tissue, human glioma sections

Negative Controls:

• DAAM2 knockdown cell lines (siRNA or shRNA-treated)

• Tissues from DAAM2 conditional knockout models (such as Olig2-cre Daam2 fl/fl mice)

• Secondary antibody-only controls for IHC/IF applications

For cancer-related studies, paired tumor and paratumor tissues provide valuable internal controls, as demonstrated in pancreatic adenocarcinoma research .

How does DAAM2 regulate oligodendrocyte differentiation and myelination?

DAAM2 critically regulates oligodendrocyte (OL) differentiation and myelination through several mechanisms:

• Cytoskeletal regulation: DAAM2 modulates the oligodendrocyte actin cytoskeleton, which is essential for proper membrane extension during myelination .

• Wnt signaling modulation: It recruits Wnt signalosome components to the cell membrane, inhibiting OL differentiation .

• Protein degradation pathways: DAAM2 facilitates ubiquitination through E3 ubiquitin ligase recruitment, affecting protein turnover during differentiation .

• Gelsolin regulation: DAAM2 induces Gelsolin ubiquitination and degradation in OLs, with Gelsolin promoting and DAAM2 inhibiting membrane spreading during late differentiation .

• PIP2 modulation: It regulates phosphatidylinositol (4,5)-bisphosphate levels at the membrane, affecting actin-binding proteins in OLs .

Studies using conditional knockout mice (Daam2 cKO) revealed that Daam2 deletion in the oligodendrocyte lineage results in myelin decompaction in both the spinal cord and corpus callosum, as well as slightly thinner myelin (higher g-ratios compared to controls) . These findings demonstrate DAAM2's essential role in forming functional myelin through cytoskeletal regulation.

What is DAAM2's role in cancer immunology, particularly in pancreatic adenocarcinoma?

Recent research has uncovered a significant role for DAAM2 in pancreatic adenocarcinoma (PAAD) immunology:

• Upregulation in tumor tissue: DAAM2 is significantly upregulated in PAAD tissues compared to paired paracancerous tissues .

• Immune microenvironment modulation: High DAAM2 expression is associated with an "immuno-hot" phenotype characterized by:

  • Increased expression of chemokines, MHC molecules, and immunomodulators

  • Higher immune and stromal scores but lower tumor purity

  • Upregulated infiltration of multiple immune cell types

• Immune checkpoint correlation: DAAM2 positively correlates with most immune checkpoints in PAAD, including PD-L1. Experimental DAAM2 knockdown significantly inhibited PD-L1 expression .

• Therapeutic response prediction: High DAAM2 expression predicted:

  • Higher responses to chemotherapy, anti-EGFR therapy, and immunotherapy

  • Lower responses to anti-ERBB2 and antiangiogenic therapy

These findings suggest DAAM2 could serve as a valuable biomarker for identifying immunologically "hot" tumors in PAAD and potentially guiding therapeutic decisions.

How does DAAM2 interact with the actin cytoskeleton and associated proteins?

DAAM2, as a formin family protein, regulates actin dynamics through interactions with several key proteins:

• Actin nucleation and elongation: DAAM2 directly regulates actin nucleation and elongation, impacting filopodia formation and cell migration .

• Rac1 interaction: In vivo screening identified Rac1 as a downstream effector of DAAM2 in oligodendrocyte cytoskeletal regulation. There is a functional relationship between DAAM2 and Rac1 during early spinal cord development .

• Gelsolin regulation: DAAM2 and Gelsolin have opposing roles in oligodendrocyte membrane spreading:

  • Gelsolin promotes membrane spreading during late differentiation

  • DAAM2 inhibits this process by inducing Gelsolin ubiquitination and degradation

  • In Daam2 cKO mice, increased Gelsolin protein levels (but not mRNA) were observed in developing white matter

• PIP5K1A interaction: During dorsal patterning of the spinal cord, DAAM2 interacts with PIP5K1A to inhibit oligodendrocyte differentiation .

• PIP2 modulation: DAAM2 regulates levels of phosphatidylinositol (4,5)-bisphosphate at the membrane, affecting downstream actin-binding proteins .

These interactions highlight DAAM2's central role in coordinating cytoskeletal dynamics across multiple cellular contexts.

What are common technical challenges when detecting DAAM2 and how can they be resolved?

When working with DAAM2 antibodies, researchers may encounter several technical challenges:

• Low signal in Western blot:

  • Solution: Use neural tissues (brain) as positive controls, as they show higher expression levels

  • Consider sample enrichment techniques for tissues with lower expression

  • Optimize protein extraction methods to preserve DAAM2 integrity, particularly given its role in cytoskeletal regulation

• Specificity concerns:

  • Solution: Validate using positive controls like mouse/rat brain tissue or transfected cells overexpressing DAAM2

  • Include knockdown/knockout samples as negative controls

  • Perform peptide competition assays to confirm specificity

• Background in IHC:

  • Solution: Start with 1:200 dilution as used in published studies (25206-1-AP, Proteintech)

  • Optimize blocking conditions and washing steps

  • Use antigen retrieval methods appropriate for formalin-fixed tissues

• Cross-reactivity with DAAM1:

  • Solution: Select antibodies specifically tested for DAAM2 specificity

  • Verify against samples with known differential expression of DAAM1 and DAAM2

What considerations are important for DAAM2 knockdown or knockout experiments?

When designing DAAM2 loss-of-function studies, consider the following:

• Knockdown approaches:

  • siRNA or shRNA targeting DAAM2 has been successfully used in cell culture models

  • Verify knockdown efficiency by both mRNA (qPCR) and protein (Western blot) analyses

  • Be aware that DAAM2 knockdown affects multiple pathways, including PD-L1 expression

• Genetic knockout models:

  • Conditional knockout approaches are preferred given DAAM2's developmental roles

  • The Olig2-cre Daam2 fl/fl (Daam2 cKO) model has been validated for studying oligodendrocyte-specific functions

  • When analyzing phenotypes, examine:

    • Myelin structure (compaction, g-ratio) using transmission electron microscopy

    • Protein levels of potential effectors like Gelsolin

    • Actin cytoskeleton dynamics

• Functional readouts:

  • For oligodendrocyte studies: membrane spreading, process extension, myelination capacity

  • For cancer studies: immune cell infiltration, checkpoint molecule expression

  • For Wnt signaling: β-catenin localization, target gene expression

• Compensatory mechanisms:

  • Consider potential compensation by DAAM1, which shares functional overlap with DAAM2 in some contexts

  • Examine downstream effectors like Rac1 and Gelsolin to determine pathway consequences

How can researchers investigate DAAM2's role in the Wnt signaling pathway?

To effectively study DAAM2's functions in Wnt signaling:

• Pathway activation assays:

  • TOP/FOP luciferase reporter assays to measure canonical Wnt activity

  • Disheveled (Dvl) complex formation analysis

  • β-catenin nuclear translocation quantification

• Protein interaction studies:

  • Co-immunoprecipitation to detect DAAM2 interactions with Wnt signalosome components

  • Proximity ligation assays to visualize protein interactions in situ

  • FRET/BRET approaches to measure dynamic interactions

• Membrane dynamics:

  • Analyze PIP2 levels at the membrane using specific biosensors

  • Assess Wnt receptor signalosomes formation through advanced microscopy

  • Examine cadherin-based junctions in the context of non-canonical Wnt signaling

• Tissue-specific contexts:

  • For dorsal spinal cord patterning: examine oligodendrocyte differentiation

  • For gut development: assess left/right asymmetry in dorsal mesentery

  • For myocardial development: evaluate sarcomere assembly

• Combined approaches:

  • Use DAAM2 overexpression and knockdown/knockout in parallel

  • Rescue experiments with DAAM2 mutants lacking specific domains

  • Pharmacological manipulation of upstream or downstream pathway components

What are emerging areas of DAAM2 research beyond current applications?

Several promising research directions for DAAM2 are emerging:

• Expanded cancer immunology applications:

  • Investigation of DAAM2 as a biomarker in cancer types beyond pancreatic adenocarcinoma

  • Development of therapeutic strategies targeting DAAM2-regulated immune pathways

  • Exploration of DAAM2's role in modulating response to immune checkpoint inhibitors

• Neurodegenerative diseases:

  • Given DAAM2's role in myelination, investigation of its potential involvement in demyelinating disorders

  • Exploration of DAAM2-targeted approaches for promoting remyelination

  • Assessment of DAAM2's contribution to glial responses in neurodegeneration

• Development of DAAM2-specific modulators:

  • Small molecules targeting DAAM2's actin regulatory functions

  • Peptide inhibitors of specific DAAM2 protein-protein interactions

  • Temporal control of DAAM2 activity in developmental contexts

• Single-cell analyses:

  • Characterization of DAAM2 expression and function at single-cell resolution

  • Integration with spatial transcriptomics to map DAAM2 activity in complex tissues

  • Temporal dynamics of DAAM2 regulation during differentiation processes

These emerging areas highlight the expanding significance of DAAM2 in both basic research and potential therapeutic applications.