DAD1 Antibody
Description
Biological Context of DAD1
DAD1 (Defender Against Cell Death 1) is a 113-amino acid protein encoded by the DAD1 gene on chromosome 14q11-q12 . It serves dual roles:
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N-linked glycosylation: As a subunit of OST, DAD1 facilitates the transfer of oligosaccharides to asparagine residues on nascent proteins in the endoplasmic reticulum (ER) .
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Anti-apoptotic activity: DAD1 inhibits programmed cell death, particularly in ER stress pathways, independent of mitochondrial or death receptor pathways . Loss of DAD1 triggers apoptosis via impaired glycosylation and ER stress, as shown in Drosophila and mammalian models .
Apoptosis and Cancer
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Prostate cancer: Elevated DAD1 expression correlates with advanced TNM and Gleason grades. DAD1 antibodies demonstrated diagnostic potential, outperforming prostate-specific antigen (PSA) in specificity .
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Therapeutic targeting: Anti-DAD1 antibodies induced cytotoxicity in cancer cells by disrupting extracellular DAD1-Fas interactions, highlighting their therapeutic utility .
Cardiomyocyte Survival
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Dad1 knockdown in neonatal rat cardiomyocytes caused apoptosis (via caspase-3 activation) and impaired cell adhesion due to defective integrin N-glycosylation . Restoring cell-matrix interactions reversed these effects .
Immune Regulation
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T cell development: Overexpression of DAD1 in transgenic mice enhanced mature T cell proliferation but did not inhibit thymocyte apoptosis during negative selection .
Mechanism of Action
DAD1 antibodies enable the study of two interconnected pathways:
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OST complex integrity: DAD1 stabilizes OST, ensuring proper glycosylation of integrins (e.g., α5/β1) and adhesion signaling .
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ER stress modulation: DAD1 loss activates PERK/ATF4 pathways, leading to JNK-mediated apoptosis .
Validation and Quality Control
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Western Blot: Detects DAD1 at 16–20 kDa in HEK-293 and HepG2 lysates .
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Immunohistochemistry: Strong staining in human liver cancer tissues, with antigen retrieval recommended .
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Cross-reactivity: Confirmed in human, mouse, and rat models .
Limitations and Future Directions
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- The expression of DAD1, DGL, and other DAD1-LIKE LIPASE (DALL) genes is induced upon wounding. DAD1 and DGL require COI1 for their expression, suggesting that a molecule other than jasmonic acid (JA) might act as a regulator of COI1. PMID: 24430866
- DAD1 participates in jasmonate formation primarily in the late wound response. PMID: 20348210
Q&A
Basic Research Questions
What experimental applications are validated for DAD1 antibodies in mammalian systems?
DAD1 antibodies are widely used in protein interaction studies and cellular localization assays. Key validated applications include:
Methodological Tip: Optimize dilutions using gradient testing and include positive controls (e.g., cancer cell lines with known DAD1 overexpression) .
How can researchers validate DAD1 antibody specificity in apoptosis studies?
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Knockdown Validation: Use siRNA targeting DAD1 in models like neonatal rat cardiomyocytes (NRCMs). Successful knockdown reduces cell viability and increases cleaved caspase-3, confirming antibody specificity .
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Functional Rescue: Overexpress DAD1 in knockdown models to reverse apoptotic phenotypes (e.g., impaired myofibrillogenesis) .
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Cross-Verification: Compare results with alternative methods (e.g., qPCR for DAD1 mRNA levels) .
Advanced Research Questions
How do contradictions in DAD1’s molecular weight arise, and how should they be resolved?
Discrepancies between observed (16–20 kDa) and calculated (12 kDa) molecular weights are linked to:
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Post-Translational Modifications: N-glycosylation via the OST complex, where DAD1 is a subunit .
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Protein Complex Association: DAD1 co-sediments with ribophorins I/II and OST48 in the endoplasmic reticulum (ER), increasing apparent size .
Resolution Strategy:
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Treat lysates with PNGase F to remove N-glycans and re-run WB .
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Use crosslinking agents (e.g., DSP) to stabilize protein complexes before IP .
What mechanisms underlie DAD1’s dual roles in apoptosis inhibition and integrin regulation?
DAD1’s anti-apoptotic function involves two pathways:
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OST-Dependent: Maintains N-glycosylation of integrins (e.g., α5/β1), preserving focal adhesion kinase (FAK) signaling and cell-matrix adhesion .
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OST-Independent: Extracellular DAD1 interacts with Fas receptor to inhibit extrinsic apoptosis in prostate cancer .
Methodological Insight: Use adhesamine or fibronectin to rescue adhesion defects in DAD1-deficient cells, distinguishing OST-dependent vs. -independent roles .
How can researchers address conflicting reports on DAD1’s role in solid vs. hematopoietic tumors?
DAD1 exhibits context-dependent expression:
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Solid Tumors: Upregulated in prostate cancer (correlates with Gleason grade) but downregulated in pancreatic SPTP .
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Hematopoietic Cancers: High variability in chronic lymphocytic leukemia, suggesting subtype-specific roles .
Experimental Design Recommendations:
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Stratify patient samples by tumor stage and subtype.
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Combine IHC with proteomics (e.g., LC-MS/MS) to map DAD1-interacting proteins in different malignancies .
What are the implications of DAD1’s conservation across species for translational research?
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Evolutionary Conservation: DAD1 homologs in Chlamys farreri (scallops) regulate apoptosis during immune responses, mirroring human roles .
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Murine Models: DAD1 knockout mice show embryonic lethality due to defective N-glycosylation, highlighting its essential role .
Cross-Species Validation Protocol:
