LPAR6 Antibody, FITC conjugated

What is LPAR6 and why is it a significant research target?

LPAR6 (Lysophosphatidic acid receptor 6), also known as P2RY5, belongs to the family of G-protein coupled receptors activated by lysophosphatidic acid (LPA). LPAR6 plays crucial roles in multiple physiological and pathological processes, particularly in hair loss mechanisms and cancer progression. Unlike LPAR1-3 (which belong to the endothelial differentiation gene/EDG family), LPAR6 is part of the non-EDG family of LPA receptors (LPAR4-6) with distinct structural and functional characteristics. Recent structural studies using cryoelectron microscopy have revealed that LPAR6 has a unique LPA binding mode that differs significantly from that of LPAR1, providing new insights into receptor-specific activation mechanisms .

What are the typical applications for LPAR6 Antibody, FITC conjugated?

LPAR6 Antibody with FITC conjugation is primarily utilized in:

• Flow cytometry/FACS: Leveraging the FITC fluorophore's excitation/emission properties (499/515 nm) with 488 nm laser lines for detection

• ELISA: For quantitative detection of LPAR6 protein

• Immunofluorescence microscopy: For cellular localization studies

The antibody enables researchers to study LPAR6 expression patterns across different cell types, particularly in cancer models and cell differentiation studies. Unlike unconjugated versions that require secondary antibody detection, the FITC conjugation provides direct visualization, reducing background and cross-reactivity concerns in multi-parameter analyses.

What are the critical storage and handling considerations for LPAR6 Antibody, FITC conjugated?

Proper storage and handling are essential for maintaining antibody functionality:

For the commercially available LPAR6-FITC antibodies, the buffer typically contains 0.01M PBS (pH 7.4), preservatives like 0.03% Proclin-300, and stabilizers such as 50% glycerol . Storage in frost-free freezers is not recommended as temperature fluctuations can damage antibody integrity .

How can I validate the specificity of LPAR6 Antibody, FITC conjugated in my experimental system?

Validation is critical to ensure your experimental results accurately reflect LPAR6 biology. A comprehensive validation approach includes:

• Positive and negative control tissues/cells: Use tissues with known LPAR6 expression patterns. Based on recent single-cell transcriptome analyses, fibro-adipogenic progenitors (FAPs), tenocytes, and certain immune cell populations (specifically monocytes and APCs) show characteristic LPAR6 expression patterns that can serve as positive controls .

• Peptide competition assay: Pre-incubate the antibody with the immunogen peptide (for many commercial LPAR6 antibodies, this is the peptide sequence from positions 292-313 AA of human LPAR6) before application to samples. Signal disappearance confirms specific binding.

• Genetic validation: Utilize LPAR6 knockout/knockdown models via CRISPR-Cas9 or RNAi approaches. Studies have demonstrated that RNAi-mediated attenuation of LPAR6 impairs HCC tumorigenicity in xenograft models , providing a research framework for such validations.

• Cross-reactivity assessment: Particularly important when studying mouse or rat models, as some antibodies raised against human LPAR6 may have predicted but unconfirmed reactivity with these species .

• Orthogonal detection methods: Compare FITC-conjugated antibody results with unconjugated antibodies in Western blot or IHC to confirm expression patterns.

How does LPAR6 expression vary across different cancer types, and what are the implications for using LPAR6 antibodies in cancer research?

LPAR6 demonstrates complex expression patterns across cancer types with significant prognostic implications:

• Breast cancer: LPAR6 is significantly downregulated in breast cancer tissues compared to normal controls. Expression is significantly higher in luminal subtypes than in HER2 and TNBC subtypes. Estrogen receptor (ER)-positive patients exhibit significantly increased LPAR6 expression compared to ER-negative patients. Interestingly, patients with higher pathological grade or clinical stage show significantly lower LPAR6 expression .

• Hepatocellular carcinoma (HCC): Contrary to breast cancer patterns, LPAR6 is commonly overexpressed in HCC specimens, and this overexpression associates with poor survival in cohort studies. Mechanistically, LPAR6 appears to drive HCC tumorigenesis through upregulation of Pim-3 via a STAT3-dependent mechanism .

These opposing expression patterns necessitate careful selection of control samples and validation in each specific cancer type. When designing experiments with LPAR6 antibodies in cancer research, consider:

• Cancer-specific expression baseline

• Correlation with clinical parameters

• Potential confounding factors (tissue heterogeneity)

• Integration with patient survival data

• Validation across multiple patient cohorts

What molecular mechanisms explain the distinct binding modes of LPA to LPAR6 compared to other LPA receptors, and how might this impact antibody epitope selection?

Recent cryoelectron microscopy studies have revealed that LPAR6 exhibits a distinctive ligand binding mode that differs significantly from that of LPAR1 (an EDG family member) . Specifically:

• LPA binding mode in LPAR6: The charged head of LPA forms an extensive polar interaction network with key polar residues on the extracellular side of transmembrane helix 5-6 and the extracellular loop 2.

• Structural distinction: This binding mode contrasts with that of LPAR1, suggesting that EDG and non-EDG families employ two distinct mechanisms for LPA binding.

• Functional implications: These structural differences may explain the varied physiological roles of different LPAR subtypes and their differential expression patterns across tissues.

When selecting antibodies, these structural insights suggest:

• Antibodies targeting extracellular loop 2 might interfere with ligand binding

• Conformational changes upon LPA binding may affect epitope accessibility

• Receptor-selective antagonists might require different design strategies for LPAR6 vs. LPAR1-3

What are the optimal dilution ranges and controls for different applications of LPAR6 Antibody, FITC conjugated?

Optimal working conditions vary by application and specific antibody manufacturer:

Note that these ranges are starting points; optimal dilutions should be determined empirically for each specific experimental system and antibody lot . For accurate results:

• Perform titration experiments to determine optimal signal-to-noise ratio

• Include appropriate blocking steps to minimize non-specific binding

• For quantitative comparisons, ensure consistent antibody lots across experiments

• Validate staining patterns with unconjugated antibodies when possible

• When analyzing tissues, account for autofluorescence with appropriate controls

How can I optimize protocols for detecting LPAR6 in different tissue and cell types given its variable expression patterns?

LPAR6 shows cell type-dependent expression patterns that require tailored protocols:

• For immune cells: LPAR6 is notably expressed in monocytes and antigen-presenting cells (APCs) . When analyzing these populations:

  • Use multi-parameter flow cytometry with lineage markers

  • Include viability dyes to exclude dead cells

  • Consider paraformaldehyde fixation for consistent results

• For skin/hair follicle studies: Given LPAR6's role in hair loss:

  • Optimize antigen retrieval methods for formalin-fixed tissues

  • Consider thick sections (>10μm) for 3D visualization

  • Counterstain with hair follicle markers for context

• For cancer tissues:

  • Account for tumor heterogeneity with single-cell approaches

  • Compare with matched normal adjacent tissue when available

  • Correlate with clinical parameters (grade, stage, ER status in breast cancer)

• General optimization strategies:

  • Adjust fixation duration based on tissue type

  • Optimize permeabilization for intracellular epitopes

  • Consider signal amplification methods for low-expressing tissues

  • Employ tissue clearing techniques for 3D visualization in complex tissues

What are the recommended approaches for multiplexing LPAR6-FITC antibody with other fluorophore-conjugated antibodies?

Effective multiplexing requires careful consideration of spectral overlap and panel design:

• Spectral considerations:

  • FITC (Ex/Em: 499/515 nm) has potential spillover into PE and other green-yellow channels

  • Compensation controls are essential for accurate data interpretation

  • Consider using alternative conjugates (e.g., AF488) for better photostability in imaging applications

• Panel design strategy:

  • Pair LPAR6-FITC with bright fluorophores (e.g., PE, APC) for less abundant targets

  • Reserve dim fluorophores for highly expressed markers

  • Include FMO (Fluorescence Minus One) controls for each channel

• Recommended marker combinations for specific applications:

  • For cancer studies: Combine with epithelial markers, proliferation markers, and immune infiltrate markers

  • For immune cell profiling: Pair with CD45, CD3, CD4/CD8, and activation markers

  • For developmental studies: Combine with lineage-specific transcription factors

• Advanced approaches:

  • Consider spectral cytometry for higher dimensional analysis

  • Implement sequential staining for challenging combinations

  • Use tyramide signal amplification for low-abundance targets

How does LPAR6 signaling differ from other LPA receptors, and what implications does this have for functional studies?

LPAR6 demonstrates unique signaling properties compared to other LPA receptors:

• G-protein coupling: Recent structural studies reveal LPAR6 can couple with both G₁₃ and Gq proteins, contributing to its diverse signaling capabilities . This coupling diversity suggests:

  • Multiple downstream pathways can be activated simultaneously

  • Different cellular responses may be context-dependent

  • Inhibitors targeting specific G-protein subtypes may have selective effects

• Structural distinctions: LPAR6 (a non-EDG family member) has a distinct LPA binding mode compared to LPAR1 (an EDG family member) :

  • The charged head of LPA forms extensive polar interactions with LPAR6

  • Key residues on transmembrane helix 5-6 and extracellular loop 2 are involved

  • These structural differences may explain differential responses to LPA across tissues

• Signaling in cancer contexts:

  • In hepatocellular carcinoma, LPAR6 drives tumorigenesis through upregulation of Pim-3 via a STAT3-dependent mechanism

  • In breast cancer, LPAR6 is regulated by miR-27a-3p, suggesting microRNA-mediated control of its expression

When designing functional studies with LPAR6 antibodies:

• Consider pathway-specific readouts to capture diverse signaling outcomes

• Include both positive and negative LPA receptor modulators as controls

• Account for potential compensatory mechanisms among LPA receptors

• Evaluate both canonical and non-canonical signaling events

What is the current understanding of LPAR6 expression patterns across different cell types and tissues based on recent single-cell transcriptome analyses?

Recent single-cell transcriptome studies have revealed detailed expression patterns of LPAR6 across diverse cell types:

• Skeletal muscle system: The Enpp2-Lpar-Plpp gene axis is dynamically expressed in skeletal muscle, with differential expression of LPAR subtypes across cell populations :

  • Fibro-adipogenic progenitors (FAPs): Express LPAR1 and LPAR4, but LPAR2, LPAR3, and LPAR5 are virtually absent

  • Tenocytes: High LPAR1 expression

  • Muscle stem cells/Satellite cells: Lower LPAR1 expression compared to tenocytes and FAPs

  • Immune cells: LPAR6 has a broader cell type-dependent expression, including in different populations of immune cells (monocytes and APCs)

  • Endothelial cells: Significant LPAR6 expression

• Cancer tissues:

  • Breast cancer: LPAR6 expression is higher in luminal subtypes compared to HER2 and TNBC subtypes, and correlates positively with ER status

  • Hepatocellular carcinoma: LPAR6 is commonly overexpressed and associates with poor survival outcomes

This heterogeneous expression pattern has important implications for research design:

• Cell type-specific functions should be evaluated rather than assuming uniform roles

• Tissue context may significantly influence LPAR6 signaling outcomes

• Single-cell approaches may be necessary to deconvolute mixed signals in heterogeneous tissues

How might LPAR6-targeted therapies be developed based on current structural and functional knowledge?

Recent structural insights into LPAR6 binding and activation open new avenues for therapeutic development:

• Structure-based drug design: The cryoelectron microscopy structure of LPA-bound human LPAR6 in complex with G proteins reveals a distinct ligand binding and recognition mode . This structural information provides:

  • Target sites for small molecule antagonists

  • Potential for receptor-selective compounds that don't affect other LPA receptors

  • Rational basis for optimizing binding affinity and specificity

• Disease-specific targeting strategies:

  • Hair loss applications: As LPAR6 mutations are associated with hypotrichosis, agonists might promote hair growth in specific contexts

  • Cancer therapeutics: Different approaches depending on cancer type:

    • For HCC, where LPAR6 is overexpressed, antagonists could disrupt STAT3-dependent Pim-3 upregulation

    • For breast cancer subtypes with altered LPAR6 expression, precision targeting based on ER status might be necessary

• Combination therapy approaches:

  • Co-targeting LPAR6 with downstream effectors like STAT3 or Pim-3

  • Combining with current standard-of-care treatments

  • Exploiting synthetic lethality with other pathways

• MicroRNA-based therapies: Given that LPAR6 is regulated by miR-27a-3p in some contexts , miRNA mimics or antagonists might offer an alternative approach to modulating LPAR6 expression.