LPAR3 Antibody

What is LPAR3 and what are its basic characteristics?

LPAR3 (also known as EDG7, LPA3, or LP-A3) is a G-protein coupled receptor that functions as a receptor for lysophosphatidic acid (LPA), a mediator of diverse cellular activities. In humans, the canonical protein has 353 amino acid residues with a molecular mass of approximately 40.1 kDa. LPAR3 is primarily localized in the cell membrane and belongs to the G-protein coupled receptor 1 protein family. This receptor undergoes post-translational modifications, including glycosylation, and is evolutionarily conserved with orthologs reported in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken species .

Which signaling pathways does LPAR3 activate?

LPAR3 primarily couples with Gαi/o and Gαq/11 heterotrimeric G proteins to facilitate various LPA-induced cellular processes. When activated, LPAR3 triggers multiple downstream signaling pathways including:

• Calcium mobilization

• Adenylyl cyclase regulation (both inhibition and activation)

• MAPK (Mitogen-Activated Protein Kinase) activation

• PLC (Phospholipase C) activation

These pathways collectively mediate diverse cellular responses including cell proliferation, survival, and migration . Research has also shown that LPAR3 activation can induce axonal branching in hippocampal cell cultures through Gq and Rho family GTPase 2 (Rnd2) signaling .

What is the tissue expression profile of LPAR3?

LPAR3 shows a tissue-specific expression pattern that varies between humans and mice:

In humans:

• Higher mRNA expression in heart, lung, pancreas, brain, testis, prostate, and ovary

In mice:

• Higher mRNA expression in testis, kidney, thymus, small intestine, lung, stomach, and brain

This differential expression profile suggests species-specific functions of LPAR3 and should be considered when designing experiments and interpreting results.

What types of LPAR3 antibodies are available for research applications?

LPAR3 antibodies are available in various formats to accommodate different experimental needs:

Researchers should select antibodies based on the specific application, target species, and required sensitivity. Polyclonal antibodies often provide broader epitope recognition, while monoclonal antibodies offer higher specificity for a single epitope .

How can I validate the specificity of an LPAR3 antibody?

Validating antibody specificity is crucial for obtaining reliable results. For LPAR3 antibodies, consider these methodological approaches:

• siRNA knockdown validation:

  • Transfect cells with LPAR3-specific siRNA (e.g., using Lipofectamine 2000)

  • Compare antibody signal between knockdown and control cells using your detection method

  • A specific antibody will show reduced signal in knockdown cells

• Overexpression validation:

  • Transfect cells with LPAR3 expression constructs

  • Compare antibody signal between overexpressing and control cells

  • A specific antibody will show increased signal in overexpressing cells

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide

  • Compare results with and without peptide competition

  • Specific binding should be blocked by the competing peptide

• Knockout/mutation models:

  • Test the antibody in LPAR3 knockout or mutant models, such as the zebrafish LPAR3 KO models described in the literature

  • Specific antibodies will show dramatically reduced or absent signal in knockout models

What considerations are important for choosing between different LPAR3 antibody products?

When selecting an LPAR3 antibody, consider these critical factors:

• Immunogen information: Verify the immunogen used (e.g., "Synthetic Peptide within Human LPAR3 aa 150-300" or specific sequences like "MNECHYDKHMDFFYNRSNTDTVDDWTGTK")

• Validated applications: Confirm the antibody has been validated for your specific application (WB, IHC, IF, etc.) and species of interest

• Citation record: Antibodies with multiple research citations often have more reliable performance data

• Predicted vs. observed band size: For Western blotting, compare the predicted size (40 kDa for LPAR3) with the observed size in validation data (e.g., 40-45 kDa)

• Cross-reactivity data: Assess if the antibody has been tested for cross-reactivity with other LPA receptor family members (LPAR1, LPAR2, etc.)

What are the optimal protocols for using LPAR3 antibodies in Western blotting?

For successful Western blot detection of LPAR3, follow these methodological guidelines:

• Sample preparation:

  • Lyse cells in appropriate buffer (e.g., Laemmli buffer with 4% SDS, 20% Glycerol, 120 mM Tris-HCl pH 6.8, 0.006% bromophenol blue, and 10% mercaptoethanol)

  • Use approximately 20-30 μg of total protein per lane

• Electrophoresis and transfer:

  • Separate proteins on 10% SDS-PAGE gels

  • Transfer to nitrocellulose membranes (semidry transfer systems work well)

• Blocking and antibody incubation:

  • Block membranes with 5% non-fat dry milk or BSA in TBST

  • Incubate with primary LPAR3 antibody at manufacturer-recommended dilution (e.g., 1:1000 for ab137497 or 2.5 μg/mL for ab23692)

  • Incubate overnight at 4°C for optimal results

• Detection considerations:

  • LPAR3 typically appears as a 40-45 kDa band

  • Multiple bands may indicate post-translational modifications

  • Use appropriate positive control cell lines (e.g., HepG2, NT2D1, or PC3 cell lysates)

How can LPAR3 antibodies be used in immunohistochemistry and immunofluorescence?

For optimal IHC and IF results with LPAR3 antibodies:

• Tissue preparation for IHC:

  • Fix tissues in 4% paraformaldehyde

  • Embed in paraffin and section at 4 μm thickness

  • Use antigen retrieval methods to expose epitopes

• Immunofluorescence protocol:

  • For cultured cells, recommended dilutions range from 0.25-2 μg/mL

  • For paraffin-embedded tissues, use 20 μg/ml concentration

• Controls and validation:

  • Include positive control tissues known to express LPAR3 (e.g., testis for human samples)

  • Include a negative control by omitting primary antibody

  • For co-localization studies, consider membrane markers to confirm LPAR3's cell membrane localization

How can functional studies of LPAR3 be conducted using antibodies?

Beyond detection, LPAR3 antibodies can be used in functional studies:

• Migration assays:

  • Use scratch wound assays to assess cell migration

  • Compare migration in cells with manipulated LPAR3 expression/activity

  • Document wound closure over time (e.g., 24 hours)

• 3D organotypic models:

  • Create three-dimensional co-culture models with fibroblasts embedded in collagen matrix

  • Seed experimental cells on top of the matrix

  • Test LPAR3 function using antibodies or LPAR3 agonists/antagonists

  • Assess phenotypes such as invasion or proliferation

• Receptor internalization studies:

  • Use fluorescently-labeled LPAR3 antibodies to track receptor internalization

  • Monitor lysosomal degradation pathway as shown in HGPS cells

What are common issues when detecting LPAR3 and how can they be resolved?

When working with LPAR3 antibodies, researchers may encounter several challenges:

• High background in Western blots:

  • Increase blocking time or blocking agent concentration

  • Reduce primary antibody concentration

  • Use higher stringency wash buffers or increase wash times

  • Consider using different secondary antibodies with lower cross-reactivity

• Multiple bands in Western blots:

  • LPAR3 can undergo post-translational modifications including glycosylation

  • Use glycosidase treatment to confirm if additional bands are due to glycosylation

  • Verify primary antibody specificity using knockout or knockdown controls

• Weak or no signal in IHC/IF:

  • Optimize antigen retrieval methods (heat-induced epitope retrieval may be necessary)

  • Adjust antibody concentration (try a range from 0.25-20 μg/mL)

  • Ensure the antibody recognizes the target species

  • Consider signal amplification methods such as tyramide signal amplification

How can I optimize LPAR3 detection in cells with low expression levels?

For detecting LPAR3 in samples with low expression:

• Increase protein loading:

  • For Western blots, increase sample loading to 40-50 μg per lane

  • Use concentration methods for dilute samples

• Signal enhancement strategies:

  • Use more sensitive detection systems (e.g., enhanced chemiluminescence plus)

  • Consider using biotin-streptavidin amplification systems

  • For IF, use high-sensitivity fluorophores and confocal microscopy

• Cell enrichment approaches:

  • Use cell sorting to enrich for LPAR3-positive populations

  • Consider using cells known to express higher levels of LPAR3 as positive controls

What is the role of LPAR3 in cancer and how can antibodies help study this connection?

LPAR3 has been implicated in several cancer types, and antibodies are essential tools for investigating these connections:

• Oral squamous cell carcinoma:

  • LPAR3 mediates LPA-stimulated migration in oral carcinoma cell lines (E10 and SCC-9)

  • This process involves PKC activity and may involve EGFR transactivation

  • LPAR3 antibodies can help assess receptor expression levels across different cancer cell lines and patient samples

• Ovarian cancer:

  • LPAR3 may play a role in ovarian cancer development

  • Antibodies can be used to compare expression patterns between normal and cancerous ovarian tissues

• Metastatic melanoma:

  • LPAR3 has been associated with B16F10 metastatic melanoma cell activity

  • LPAR1/3 antagonists suggest LPAR3's involvement in metastatic processes

Research methodology should include:

• Expression analysis using IHC and Western blotting to correlate LPAR3 levels with disease progression

• Functional studies using LPAR3 knockdown or overexpression combined with cell migration, invasion, and proliferation assays

• Signaling pathway analysis to determine cancer-specific LPAR3 mechanisms

How is LPAR3 involved in neurodevelopmental processes and neurological disorders?

LPAR3 plays several roles in neuronal development and function:

• Axonal branching:

  • LPAR3 activation induces axonal branching in hippocampal cell cultures

  • This process is mediated through Gq and Rho family GTPase 2 (Rnd2)

• Behavior regulation:

  • LPAR3 knockout zebrafish display behavioral abnormalities including:

    • Hyperactivity-like behavior in larvae

    • Altered exploratory behavior

    • Impaired social interaction

    • Disrupted circadian rhythm locomotor activity

    • Memory deficiency in passive avoidance tests

• Central post-stroke pain:

  • LPAR3, along with LPAR1, mediates the development of central post-stroke pain (CPSP)

Research approaches should include:

• Behavioral testing in LPAR3 knockout or knockdown models

• Immunohistochemical analysis of LPAR3 expression in different brain regions

• Electrophysiological studies of neuronal activity in relation to LPAR3 signaling

What is the connection between LPAR3 and premature aging conditions?

Recent research has uncovered an intriguing link between LPAR3 and premature aging:

• Hutchinson-Gilford progeria syndrome (HGPS):

  • LPAR3 protein levels are downregulated in HGPS through internalization and lysosomal degradation

  • LPAR3 activation increases expression of antioxidant enzymes

  • LPAR3 helps inhibit reactive oxygen species (ROS) accumulation and ameliorates cell senescence

• Zebrafish aging model:

  • LPAR3 deficiency in zebrafish causes premature aging phenotypes in multiple organs

  • LPAR3-deficient zebrafish have shorter lifespans

  • This suggests LPAR3 plays a key role in preventing premature aging

Research methodologies should include:

• Analysis of LPAR3 levels in aging tissues using antibodies

• ROS measurement assays in cells with manipulated LPAR3 expression

• Senescence markers assessment (e.g., β-galactosidase staining, p16 expression)

• Lifespan studies in model organisms with LPAR3 mutations

How can siRNA and CRISPR/Cas9 techniques be combined with LPAR3 antibodies for comprehensive studies?

Integrating genetic manipulation with antibody-based detection provides powerful insights into LPAR3 function:

• siRNA knockdown methodology:

  • Transfect cells with LPAR3-specific siRNA (e.g., ON-TARGET plus siRNA at 60 nM final concentration)

  • Use transfection reagents like Lipofectamine 2000 (3 μl per well in a 12-well plate)

  • Include non-targeting siRNA controls

  • Culture transfected cells for 72 hours before proceeding with experiments

  • Confirm knockdown efficiency using LPAR3 antibodies in Western blotting or qRT-PCR

• CRISPR/Cas9 knockout approach:

  • Design guide RNAs targeting exons of the LPAR3 gene

  • Create stable knockout cell lines

  • Validate knockout efficiency using LPAR3 antibodies

  • Perform phenotypic and functional assays to assess LPAR3's role

• Integrated validation:

  • Compare results between transient knockdown and stable knockout models

  • Use LPAR3 antibodies to confirm protein absence in knockout models

  • Combine with rescue experiments by reintroducing LPAR3 expression

What advanced imaging techniques can be used with LPAR3 antibodies to study receptor dynamics?

Advanced imaging techniques provide insights into LPAR3 trafficking and interactions:

• Live-cell imaging of LPAR3:

  • Use fluorescently-tagged LPAR3 antibodies or LPAR3-GFP fusion proteins

  • Monitor receptor internalization and recycling in real-time

  • Track LPAR3 movement in response to ligand stimulation

• Super-resolution microscopy:

  • Techniques like STORM, PALM, or STED can resolve LPAR3 localization at nanometer resolution

  • Visualize LPAR3 clustering and organization within membrane microdomains

  • Combine with other membrane markers to study co-localization

• FRET/BRET approaches:

  • Study LPAR3 protein-protein interactions using Förster resonance energy transfer

  • Investigate proximity between LPAR3 and downstream signaling partners

  • Monitor conformational changes upon receptor activation

How can phospho-specific antibodies complement LPAR3 research?

Phospho-specific antibodies targeting LPAR3 downstream effectors provide valuable insights:

• Signaling pathway analysis:

  • Use phospho-specific antibodies against ERK1/2 (Thr202/Tyr204), p38 (Thr180/Tyr182), and Akt (Ser473) to track LPAR3-mediated signaling

  • Perform Western blots or capillary isoelectric focusing to detect activation states

• Temporal dynamics:

  • Conduct time-course experiments following LPAR3 activation

  • Use phospho-antibodies to map the sequence of signaling events

  • Determine how different LPA species affect signaling dynamics

• Single-cell analysis:

  • Combine phospho-specific antibodies with flow cytometry or mass cytometry

  • Identify heterogeneity in LPAR3 signaling within cell populations

  • Correlate LPAR3 expression levels with downstream signaling activation