Aplnr Antibody

What is APLNR and why is it significant in research?

APLNR, also known as APJR, APJ, AGTRL1, HG11, or the APJ receptor, is a G protein-coupled receptor (GPCR) with a molecular weight of approximately 42.7 kilodaltons . This receptor has gained significant attention due to its involvement in multiple physiological and pathological processes.

APLNR's significance in research stems from its crucial roles in:

• Angiogenesis and vascular development

• Cancer progression, particularly in glioblastoma (GBM)

• Cardiovascular regulation

• Neural signaling pathways

Research has demonstrated that both APLNR and its ligand, apelin (APLN), are upregulated in various cancers, particularly in GBM where they control tumor cell invasiveness and angiogenesis . Moreover, APLNR has emerged as an essential gene for cancer immunotherapy . The receptor's multi-faceted roles make APLNR antibodies valuable tools for investigating disease mechanisms and developing targeted therapeutics.

What applications are APLNR antibodies commonly used for?

APLNR antibodies are versatile tools employed in various experimental techniques. Based on available product information, researchers utilize these antibodies primarily in:

When selecting applications, researchers should consider that APLNR is detected in various tissues, with notable expression in tumor vasculature, GBM pseudopalisades, neurons, and astrocytes .

How should I select the appropriate APLNR antibody for my research?

The selection of an appropriate APLNR antibody is critical for experimental success. Consider these methodological aspects:

• Target Specificity: Verify whether the antibody recognizes specific regions (N-terminal, C-terminal, or internal domains) of APLNR. Different epitopes may be more accessible depending on experimental conditions.

• Species Reactivity: Commercial APLNR antibodies show varying cross-reactivity across species. Based on available products, antibodies with human reactivity are most common, followed by those recognizing mouse and rat APLNR . For comparative studies, select antibodies that recognize orthologous regions.

• Validation Data: Prioritize antibodies with extensive validation data relevant to your application. Look for publications demonstrating successful use in your specific experimental context.

• Clonality:

  • Monoclonal antibodies offer high specificity for a single epitope but may be sensitive to conformational changes

  • Polyclonal antibodies recognize multiple epitopes, increasing detection sensitivity but potentially decreasing specificity

• Application-Specific Optimization: Different applications require antibodies with distinct properties. For example, antibodies suitable for Western blot may not perform optimally in immunohistochemistry due to differences in protein conformation.

What controls should I include when using APLNR antibodies?

Proper controls are essential for interpreting results from experiments using APLNR antibodies:

• Positive Control: Include tissues or cell lines known to express APLNR, such as:

  • GBM tissue sections or cell lines (high APLNR expression)

  • Vascular endothelial cells from tumor samples

  • Hypoxic tissue regions (APLNR is co-expressed with VEGFA in hypoxic areas)

• Negative Control:

  • Primary antibody omission

  • Isotype control antibody

  • Tissues from APLNR knockout models

  • Cells with APLNR knockdown via shRNA

• Peptide Competition: Pre-incubate the APLNR antibody with a blocking peptide corresponding to the immunogen to confirm binding specificity.

• Signal Specificity Controls: For co-localization studies, include single-labeled controls to assess bleed-through and non-specific binding.

How can I validate APLNR antibody specificity?

Methodological approaches to validate APLNR antibody specificity include:

• Western Blot Analysis: Confirm the presence of a band at the expected molecular weight (approximately 42.7 kDa) . Multiple bands may indicate splice variants, post-translational modifications, or non-specific binding.

• Genetic Validation:

  • Compare staining patterns in wild-type versus APLNR knockout tissues

  • Use siRNA or shRNA knockdown models to confirm signal reduction

• Orthogonal Verification: Compare protein detection with mRNA expression data using techniques such as RT-PCR or RNA sequencing.

• Cross-Validation: Use multiple antibodies targeting different epitopes of APLNR and compare staining patterns.

• Peptide Competition Assay: Pre-incubation with the immunizing peptide should eliminate specific binding.

How can I use APLNR antibodies to study receptor signaling bias?

APLNR exhibits biased signaling, where different ligands or mutations can preferentially activate G-protein or β-arrestin pathways. Recent structural insights have revealed mechanisms underlying this signaling bias .

Methodological Approach:

• Differential Detection of Activated States: Use conformation-specific antibodies that recognize active versus inactive receptor states, or G-protein-coupled versus β-arrestin-coupled states.

• Co-Immunoprecipitation Studies:

  • Use APLNR antibodies to pull down receptor complexes

  • Probe for associated signaling proteins (Gi proteins versus β-arrestins)

  • Compare binding patterns after stimulation with balanced agonists (e.g., apelin-13) versus biased agonists (e.g., MM07, CMF-019, or WN561)

• Immunofluorescence Approaches:

  • Monitor receptor internalization patterns (indicative of β-arrestin recruitment)

  • Compare localization after treatment with balanced versus G-protein-biased agonists

  • Analyze colocalization with downstream signaling components

• Key Residues for Bias Analysis: Focus on specific amino acid residues identified as critical for signaling bias, including:

  • W85, Y88, and F257, which are involved in G-protein-biased signaling

  • Residues that differ in interactions between balanced agonists (cmpd644) and G-protein-biased agonists (CMF-019)

These approaches can help determine how structural modifications of APLNR ligands affect signaling bias, potentially guiding the development of more specific therapeutic agents with reduced side effects.

What are the best approaches for studying APLNR expression in tumor vasculature?

APLNR expression is dramatically upregulated in tumor-associated vasculature, particularly in GBM microvascular proliferations . For comprehensive analysis:

Methodological Workflow:

• Multiplex Immunofluorescence:

  • Co-stain with APLNR antibodies and endothelial markers (CD31, CD34)

  • Include pericyte markers (α-SMA, PDGFR-β) to assess vessel maturity

  • Add hypoxia markers (CAIX, HIF-1α) to correlate with microenvironmental conditions

• Quantitative Analysis:

  • Measure vessel density, branching, and diameter in APLNR-positive versus negative regions

  • Quantify APLNR expression intensity relative to vessel morphology

  • Analyze spatial relationship between APLNR expression and pseudopalisading necrosis in GBM

• Serial Xenograft Models:

  • Monitor changes in APLNR expression during the angiogenic switch in GBM using serial xenograft models

  • Compare invasive versus angiogenic GBM phenotypes

  • Use lineage tracing with APLN-creER and APLNR-creER systems for temporal studies

• Functional Manipulation:

  • Compare vessel phenotypes after APLNR antagonist treatment (e.g., Apelin-F13A, MM54)

  • Analyze effects of combined VEGF and APLNR inhibition

  • Evaluate vascular normalization parameters after APLNR modulation

This comprehensive approach enables detailed characterization of APLNR's role in tumor angiogenesis and identification of potential therapeutic vulnerabilities.

How can I optimize APLNR antibody detection in neural tissues?

APLNR is expressed in neurons and astrocytes, requiring specific considerations for detection in neural tissues :

• Tissue Preparation:

  • Use fresh-frozen sections for optimal epitope preservation

  • For fixed tissues, test multiple antigen retrieval methods (heat-induced versus enzymatic)

  • Consider short fixation times to prevent epitope masking

• Multi-Labeling Strategies:

  • Combine APLNR antibodies with neuronal markers (NeuN, MAP2)

  • Co-stain with astrocyte markers (GFAP, S100B)

  • Include BBB markers (Claudin-5, ZO-1) to study APLNR in neurovascular interactions

• Regional Analysis:

  • Focus on hippocampal regions with documented APLNR expression

  • Examine areas of reactive astrogliosis surrounding tumors

  • Compare expression in normal versus pathological neural tissues

• Functional Correlation:

  • Relate APLNR expression to astrocyte maturation and BBB integrity

  • Examine APLNR distribution in the context of protective astroglial barriers

  • Analyze APLNR expression during wound healing processes in neural tissues

This methodological approach enables detailed characterization of APLNR in the complex cellular environment of neural tissues and tumors.

How can I use APLNR antibodies to evaluate therapeutic efficacy?

APLNR-targeted therapeutics include signaling-biased agonists, antagonists, and blocking antibodies . To evaluate their efficacy:

• Receptor Occupancy Assays:

  • Use fluorescently-labeled APLNR antibodies to quantify receptor availability before and after therapeutic treatment

  • Compete labeled antibodies with unlabeled therapeutics to determine binding kinetics

• Downstream Signaling Analysis:

  • Monitor G-protein activation after treatment with biased agonists like MM07, CMF-019, or WN561

  • Compare receptor internalization patterns between balanced and G-protein-biased agonists

  • Assess β-arrestin recruitment in the presence of APLNR modulators

• Functional Outcomes Assessment:

  • Quantify anti-angiogenic effects in tumor models after APLNR antagonist (Apelin-F13A, MM54) treatment

  • Measure changes in tumor cell invasion following therapeutic intervention

  • Analyze combined VEGFA blockade and APLNR inhibition effects

• Therapeutic Resistance Monitoring:

  • Track changes in APLNR expression levels and localization during treatment

  • Identify compensatory signaling pathways activated after APLNR blockade

  • Analyze receptor mutations or variants that emerge following therapeutic pressure

These methodologies provide comprehensive evaluation of therapeutic efficacy and potential resistance mechanisms in APLNR-targeted interventions.

What are common pitfalls in APLNR antibody experiments and how can I avoid them?

• Non-specific Binding:

  • Issue: Multiple bands or diffuse staining patterns

  • Solution: Optimize antibody concentration, increase blocking duration, and use more stringent washing procedures

• Inconsistent Results Between Applications:

  • Issue: An antibody works for Western blot but not immunohistochemistry

  • Solution: Different applications expose different epitopes; select application-specific validated antibodies

• Species Cross-Reactivity Limitations:

  • Issue: Limited reactivity across species despite sequence homology

  • Solution: Verify epitope conservation across species and select broadly reactive antibodies for comparative studies

• Receptor Conformational Changes:

  • Issue: Reduced detection after ligand binding or signaling activation

  • Solution: Use multiple antibodies targeting different epitopes or fixed versus non-fixed preparations

How can I quantitatively analyze APLNR expression in research samples?

For rigorous quantitative analysis of APLNR expression:

• Semi-quantitative Western Blot Analysis:

  • Normalize APLNR band intensity to housekeeping proteins

  • Use standard curves with recombinant APLNR for absolute quantification

  • Include gradient loading to ensure detection within linear range

• Immunofluorescence Quantification:

  • Employ automated image analysis software for unbiased quantification

  • Include calibration standards in each experiment

  • Report relative fluorescence units or integrated density values

• Receptor Density Measurements:

  • Use saturation binding assays with labeled ligands or antibodies

  • Calculate Bmax values for receptor density

  • Compare results across experimental conditions or disease states

• Single-Cell Analysis:

  • Combine APLNR antibodies with flow cytometry or CyTOF for population analysis

  • Apply spatial transcriptomics with in situ hybridization to correlate mRNA and protein levels

  • Implement multiplexed ion beam imaging for high-dimensional analysis

These quantitative approaches enable robust comparative analysis of APLNR expression across experimental conditions.