ADGRL4 Antibody

What is ADGRL4 and why is it a significant research target?

ADGRL4, also known as ELTD1 or latrophilin-2, is an orphan adhesion G protein-coupled receptor (aGPCR) primarily expressed in endothelial cells and smooth muscle cells. It has emerged as an important research target due to its critical role in regulating both physiological and tumor angiogenesis, making it relevant for cancer research and vascular biology . ADGRL4 functions as a cell adhesion molecule involved in neuronal development and synaptic plasticity, playing a key role in regulating synaptic adhesion and neurotransmitter release . This multi-functional receptor is located on human chromosome 1p31.1 and produces a protein of approximately 77.8 kDa .

ADGRL4 expression is induced by VEGFR and TGF-β signaling while being repressed by DLL4, positioning it within critical angiogenic regulatory networks . Recent research has demonstrated that ADGRL4 silencing affects the Notch signaling pathway by upregulating DLL4 and suppressing JAG1 and HES2, indicating its importance in endothelial differentiation and homeostasis . Additionally, ADGRL4 has been implicated in regulating metabolic processes within endothelial cells, with silencing studies showing impacts on expression of metabolic enzymes like ATP Citrate Lyase (ACLY) and the mitochondria-to-cytoplasm citrate transporter (SLC25A1) .

What should researchers consider when selecting an ADGRL4 antibody for experimental use?

When selecting an ADGRL4 antibody for research applications, several critical factors must be evaluated to ensure experimental success. First, researchers should consider target species compatibility. For example, the ADGRL4 Rabbit Polyclonal Antibody (CAB14595) demonstrates reactivity against human, mouse, and rat ADGRL4 proteins, making it suitable for cross-species studies . This broad reactivity is particularly valuable for translational research comparing animal models to human specimens.

The immunogen composition is another crucial consideration. For instance, the CAB14595 antibody was generated using a recombinant fusion protein containing a sequence corresponding to amino acids 110-406 of human ADGRL4 (NP_071442.2) . Understanding the specific epitope region helps predict potential cross-reactivity issues and explains why some antibodies may recognize certain protein domains but not others. Researchers should verify whether the antibody recognizes the extracellular domain, transmembrane regions, or intracellular portions of ADGRL4.

Antibody validation is essential for ensuring specificity. Reputable suppliers validate their ADGRL4 antibodies on tissues known to express or not express ADGRL4 . Researchers should review validation data showing testing on positive tissues (such as endothelial cells) and negative controls. Additionally, examine whether validation has been performed specifically for your intended application (Western blot, immunohistochemistry, flow cytometry, etc.), as performance can vary significantly between applications.

Finally, consider antibody format and concentration. ADGRL4 antibodies are typically available in different sizes (e.g., 20μL, 100μL) and may have specific storage requirements . For quantitative applications, understanding the antibody concentration and recommended dilution ranges is essential for experimental standardization and reproducibility.

What are optimal protocols for detecting ADGRL4 in Western blot applications?

Optimizing Western blot protocols for ADGRL4 detection requires careful consideration of this protein's properties as a multi-pass membrane receptor. Sample preparation is particularly critical for membrane proteins like ADGRL4. Use RIPA buffer supplemented with protease inhibitors for efficient extraction, but avoid extended boiling as this may cause aggregation of transmembrane proteins. Instead, heat samples at 70°C for 10 minutes to maintain protein integrity while ensuring denaturation .

For gel electrophoresis, use 8-10% SDS-PAGE gels for optimal resolution of ADGRL4, which has a molecular weight of approximately 77.8 kDa . Higher percentage gels may impede the migration of this relatively large membrane protein. Load adequate protein (40-60 μg of total protein) to ensure detection, as ADGRL4 may be expressed at relatively low levels in some cell types compared to housekeeping proteins.

The transfer step is crucial for membrane proteins. Perform wet transfer at lower voltage (30V) overnight at 4°C rather than rapid high-voltage transfer, as this improves the efficiency of transferring membrane proteins to the PVDF or nitrocellulose membrane. For antibody incubation, block with 5% non-fat dry milk or BSA in TBST for at least 1 hour at room temperature to reduce background. When using ADGRL4 Rabbit Polyclonal Antibody (CAB14595), follow the manufacturer's recommended dilution, typically in the range of 1:500-1:2000 .

For detection, use enhanced chemiluminescence (ECL) systems, and consider more sensitive detection methods such as ECL Plus for low-abundance samples. The expected band for ADGRL4 should appear at approximately 77.8 kDa, though variations may occur due to post-translational modifications, particularly glycosylation . Always include positive controls from tissues known to express ADGRL4, such as endothelial cells, to confirm successful detection.

How can researchers effectively validate ADGRL4 antibody specificity?

Validating ADGRL4 antibody specificity is essential for generating reliable research data. Multiple complementary approaches should be employed to ensure comprehensive validation. First, implement peptide competition assays by pre-incubating the ADGRL4 antibody with the specific immunogenic peptide used to generate it (in the case of CAB14595, this would involve amino acids 110-406 of human ADGRL4) . This competition should abolish or significantly reduce specific staining in Western blots, immunohistochemistry, or flow cytometry.

Second, utilize ADGRL4 knockdown or knockout controls. Perform siRNA-mediated silencing of ADGRL4 in appropriate cell models, such as HUVECs, which naturally express ADGRL4 . Compare antibody detection between wild-type and ADGRL4-silenced cells to confirm signal reduction proportional to the knockdown efficiency. This approach provides functional validation of antibody specificity within biological systems.

Third, verify tissue expression patterns. Test the antibody on multiple tissues with known ADGRL4 expression profiles. Strong signals should be detectable in endothelial cells and vascular smooth muscle cells, while tissues with minimal expression should show correspondingly weak signals . Compare antibody-based detection patterns with published mRNA expression data from resources like GTEx or Human Protein Atlas to ensure concordance.

Finally, compare multiple antibodies targeting different epitopes of ADGRL4. If different antibodies raised against distinct regions of ADGRL4 show similar detection patterns, this provides additional confidence in specificity. Document and report all validation steps in publications to increase the reproducibility and reliability of ADGRL4 research findings.

What are the common challenges in immunohistochemical detection of ADGRL4?

Immunohistochemical (IHC) detection of ADGRL4 presents several technical challenges that require methodological optimization. The first major challenge involves epitope masking, which occurs frequently with transmembrane proteins like ADGRL4. The protein's complex structure with multiple domains can result in epitope inaccessibility during fixation and processing. To overcome this limitation, researchers should test multiple antigen retrieval methods, comparing both heat-induced epitope retrieval using citrate buffer (pH 6.0) and Tris-EDTA buffer (pH 9.0) . Additionally, comparing different fixation protocols may be necessary, as overfixation can permanently mask epitopes relevant for ADGRL4 detection.

Signal specificity represents another significant challenge. ADGRL4 expression in endothelial cells may be confused with general vascular staining or with other vascular markers. To ensure specificity, researchers must perform rigorous controls including: isotype controls to assess non-specific binding; positive controls using tissues known to express ADGRL4 (such as placental tissue); negative controls using tissues where ADGRL4 expression is minimal; and peptide competition assays to confirm antibody specificity . Additionally, dual staining with established endothelial markers like CD31 can help confirm the vascular localization of ADGRL4 signals.

Background staining often complicates ADGRL4 detection, particularly in vascular-rich tissues. To mitigate this, optimize blocking protocols by using species-specific serum (5-10%) combined with protein blockers like BSA. The inclusion of 0.1-0.3% Triton X-100 in blocking solutions can improve antibody penetration while reducing non-specific binding. Furthermore, careful titration of primary antibody concentration is essential to determine the optimal signal-to-noise ratio for each specific tissue type.

Lastly, variability in ADGRL4 expression levels across different vascular beds and in pathological conditions can complicate standardization of detection protocols. Researchers should develop tailored protocols for specific research questions, potentially adjusting antibody concentrations and detection systems based on the expected expression levels in their experimental system.

How can I use ADGRL4 antibodies to investigate the role of this receptor in angiogenesis?

Investigating ADGRL4's role in angiogenesis requires sophisticated experimental approaches combining antibody-based detection with functional assays. ADGRL4 antibodies can be employed in multiple complementary strategies to elucidate its specific contributions to angiogenic processes. First, researchers should establish baseline expression patterns in relevant models. Immunofluorescence staining of developing vascular networks in embryoid bodies, retinal explants, or tumor sections can reveal ADGRL4 localization during active angiogenesis . Co-staining with markers for endothelial tip cells (ESM1, ANGPT2) versus stalk cells (NOTCH1, JAG1) can clarify ADGRL4's distribution in these functionally distinct endothelial subpopulations.

For manipulation studies, combine ADGRL4 antibodies with functional knockdown experiments. After siRNA-mediated silencing of ADGRL4 in endothelial cells, use antibodies to confirm knockdown efficiency at the protein level before proceeding with functional angiogenesis assays . This validation step is crucial as mRNA reduction doesn't always translate to proportional protein reduction due to variations in protein half-life or post-transcriptional regulation.

To investigate signaling mechanisms, use phospho-specific antibodies against downstream effectors following ADGRL4 manipulation. Research has shown that ADGRL4 silencing affects Notch signaling components, upregulating DLL4 while suppressing JAG1 and HES2 . Monitoring these changes with specific antibodies during different stages of angiogenesis can reveal temporal dynamics of signaling pathway crosstalk.

For therapeutic development, ADGRL4 antibodies can be used to assess whether pharmacological compounds affect receptor expression or localization. Since ADGRL4 regulates tumor angiogenesis, using antibodies to quantify its expression before and after treatment with candidate drugs may identify compounds that modulate this pathway. Additionally, neutralizing antibodies against ADGRL4's extracellular domain could be developed and tested for their ability to block angiogenic functions, potentially offering new therapeutic strategies for pathological angiogenesis.

What approaches can detect interactions between ADGRL4 and the Notch signaling pathway?

Investigating interactions between ADGRL4 and the Notch signaling pathway requires multifaceted experimental approaches that can detect regulatory relationships at transcriptional, protein, and functional levels. Research has established that ADGRL4 silencing affects Notch pathway components, specifically upregulating DLL4 while suppressing JAG1 and HES2 expression . To comprehensively characterize these interactions, researchers should implement a combination of protein-level, genetic, and functional methodologies.

At the protein level, co-immunoprecipitation experiments can reveal physical associations between ADGRL4 and Notch pathway components. Using validated ADGRL4 antibodies, researchers can immunoprecipitate protein complexes from endothelial cells and probe for Notch receptors, ligands, or downstream effectors. Complementary approaches include proximity ligation assays, which can detect protein interactions in situ with high sensitivity. For these studies, careful antibody validation is essential, as nonspecific binding could lead to false-positive results. Controls should include immunoprecipitation with isotype-matched irrelevant antibodies and validation in ADGRL4-silenced cells.

Genetic manipulation provides another powerful approach. Sequential and simultaneous silencing experiments can establish epistatic relationships between ADGRL4 and Notch pathway genes. For example, researchers could silence ADGRL4 followed by DLL4 silencing to determine whether the phenotypic effects of ADGRL4 knockdown are mediated through DLL4 upregulation. Similarly, constitutive activation of Notch signaling through NICD overexpression can be combined with ADGRL4 manipulation to assess pathway hierarchy. Western blotting with specific antibodies against ADGRL4, DLL4, JAG1, and HES2 provides crucial validation for these genetic studies .

Functional readouts are essential for establishing biological relevance. Endothelial sprouting assays with combined manipulation of ADGRL4 and Notch components can reveal functional interactions. Since ADGRL4 regulates endothelial tip cell behavior, quantitative analysis of tip cell formation under conditions of ADGRL4 silencing, Notch inhibition (DAPT treatment), or combined intervention provides insights into pathway crosstalk. Immunofluorescence staining for ADGRL4 and Notch pathway components in these sprouting structures can reveal spatial relationships during active angiogenesis.

How can researchers investigate the impact of ADGRL4 on endothelial cell metabolism?

Investigating ADGRL4's impact on endothelial metabolism requires an integrated approach combining targeted analysis of specific metabolic pathways with global metabolic profiling. Research has shown that ADGRL4 silencing induces expression of the cytoplasmic metabolic regulator ATP Citrate Lyase (ACLY) and the mitochondria-to-cytoplasm citrate transporter (SLC25A1) . Additionally, metabolomic analysis has implicated ADGRL4 in pyrimidine, amino acid, and sugar metabolism, with significant alterations in metabolites including cis-aconitic acid, UDP-glucoronate, and fructose 2,6-diphosphate following ADGRL4 silencing .

To comprehensively characterize these metabolic effects, researchers should first verify changes in key metabolic enzymes using ADGRL4 antibodies in combination with antibodies against metabolic regulators. Western blot analysis comparing control and ADGRL4-silenced endothelial cells should quantify expression changes in ACLY, SLC25A1, and other metabolic enzymes. Phospho-specific antibodies can further reveal activation states of these enzymes, as metabolic regulation often occurs through post-translational modifications rather than expression changes alone.

Metabolic flux analysis provides deeper insights into pathway activities beyond static measurements. Researchers should perform stable isotope tracing studies using 13C-labeled glucose, glutamine, or acetate in ADGRL4-manipulated endothelial cells. This approach can determine whether ADGRL4 impacts substrate utilization preferences or specific branch points in central carbon metabolism. Mass spectrometry analysis of labeled metabolites will reveal how carbon atoms flow through metabolic pathways differently when ADGRL4 levels are altered.

Bioenergetic profiling using technologies like Seahorse extracellular flux analysis can quantify how ADGRL4 manipulation affects cellular respiration and glycolysis. By measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in control versus ADGRL4-silenced or overexpressing cells, researchers can determine whether ADGRL4 regulates the balance between oxidative phosphorylation and glycolysis—a critical aspect of endothelial metabolic adaptation during angiogenesis.

Finally, functional metabolic assays should assess how ADGRL4-induced metabolic changes impact endothelial behaviors. These include measurements of glucose uptake using fluorescent glucose analogs, fatty acid oxidation using radiolabeled substrates, and de novo lipogenesis rates. Correlating these functional readouts with angiogenic phenotypes can establish causal relationships between ADGRL4-regulated metabolism and endothelial function.

What strategies can identify potential ligands for the orphan receptor ADGRL4?

Identifying ligands for orphan receptors like ADGRL4 represents one of the most challenging aspects of GPCR research. As a member of the adhesion GPCR family, ADGRL4 possesses a large extracellular domain that likely interacts with multiple partners, complicating ligand discovery efforts . A comprehensive ligand identification strategy must combine computational predictions, biochemical screening, and functional validation approaches.

Computational prediction approaches provide an efficient starting point. Researchers should perform structural homology modeling of ADGRL4's extracellular domain based on crystallized structures of related adhesion GPCRs. This in silico model can then be used for molecular docking simulations with candidate ligands, especially extracellular matrix proteins and growth factors involved in angiogenesis. Additionally, analyzing expression correlation databases can identify proteins whose expression patterns closely match ADGRL4 across tissues, potentially revealing physiologically relevant interaction partners.

Direct binding assays represent the gold standard for ligand identification. Researchers should develop recombinant protein constructs comprising ADGRL4's extracellular domain fused to Fc or His tags for purification. These purified proteins can then be used in surface plasmon resonance (SPR) or biolayer interferometry assays to screen for binding partners. Candidate libraries should include vascular basement membrane components, angiogenic growth factors, and cell surface proteins expressed on adjacent cells in the vascular niche. ADGRL4 antibodies can serve as positive controls for binding assays and help validate the proper folding of recombinant ADGRL4 constructs.

Cell-based screening approaches offer a complementary strategy. Researchers can develop ADGRL4-overexpressing reporter cell lines with fluorescent or luminescent readouts tied to pathway activation. These engineered cells can then be exposed to tissue extracts, conditioned media from various cell types, or fractionated protein libraries to identify activating components. Once candidate activators are identified, ADGRL4 antibodies can be used to confirm specific binding and to potentially block the interaction as a validation step.

How can ADGRL4 antibodies be used to study its role in pathological conditions?

ADGRL4 antibodies provide powerful tools for investigating this receptor's involvement in pathological conditions, particularly in cancer angiogenesis where ADGRL4/ELTD1 has been identified as a key regulator . Research has shown elevated ADGRL4 expression in tumor-associated endothelial cells across multiple cancer types, suggesting its importance in tumor vascularization . Antibody-based approaches can elucidate both basic mechanisms and potential therapeutic applications in these contexts.

For diagnostic and prognostic applications, immunohistochemical analysis using validated ADGRL4 antibodies can quantify expression patterns in patient tumor samples. Researchers should develop standardized scoring systems based on staining intensity and distribution within the tumor vasculature. Digital pathology approaches using automated image analysis can provide objective quantification. Correlation of ADGRL4 expression patterns with clinical outcomes, treatment responses, and other angiogenic markers can reveal its value as a biomarker. This requires careful validation of antibody specificity in tumor tissues, where non-specific binding can be problematic due to areas of necrosis and high protein content.

Mechanistic studies in disease models benefit from multiple antibody applications. Dual immunofluorescence staining with ADGRL4 antibodies alongside markers for endothelial subtypes (tip vs. stalk cells), pericyte coverage, or basement membrane components can reveal how ADGRL4 influences vascular structure and maturation in pathological settings. In situ proximity ligation assays can detect protein-protein interactions between ADGRL4 and other signaling components within the diseased tissue microenvironment, providing spatial context that is lost in biochemical assays.

For therapeutic development, neutralizing antibodies targeting ADGRL4's extracellular domain represent a potential intervention strategy. Researchers can generate monoclonal antibodies against specific epitopes of ADGRL4 and test their ability to block angiogenic functions in vitro and in vivo. Flow cytometry using these antibodies can confirm target engagement on endothelial cells, while functional assays (tube formation, sprouting, migration) can assess their impact on angiogenic processes. In animal models, antibody treatment efficacy can be monitored through non-invasive imaging of tumor vasculature followed by immunohistochemical analysis of vascular density and morphology.

Monitoring treatment responses provides another important application. In preclinical models and potentially clinical trials, ADGRL4 antibodies can assess whether established therapies (anti-angiogenic drugs, chemotherapy, radiation) alter ADGRL4 expression as part of their mechanism or as a resistance response. Sequential biopsies analyzed by immunohistochemistry or circulating endothelial cells analyzed by flow cytometry can provide this information, potentially identifying ADGRL4 as a response biomarker.