FARP2 Antibody

What is FARP2 and what cellular functions does it regulate?

FARP2 is a guanine nucleotide exchange factor (GEF) belonging to the Dbl family that contains a 4.1, ezrin, radixin and moesin (FERM) domain, a Dbl-homology (DH) domain, and two pleckstrin homology (PH) domains . FARP2 primarily functions as a GEF for Rho family GTPases, particularly Cdc42 and possibly Rac1, though there are conflicting reports about substrate specificity .

FARP2 plays critical roles in several cellular processes:

• Regulation of cell polarity and tight junction formation through interactions with aPKCι

• Neuronal axon guidance and dendrite outgrowth via plexin signaling pathways

• Bone homeostasis regulation

• Participation in feedback control mechanisms for polarity establishment

The protein adopts an auto-inhibited conformation in which the GEF substrate-binding site is blocked by the last helix in the DH domain and the two PH domains, providing a regulatory mechanism for its activity .

How is FARP2 activity regulated in cells?

FARP2 activity is regulated through multiple mechanisms:

• Auto-inhibition: Crystal structures reveal that FARP2 adopts an auto-inhibited conformation where its GEF substrate-binding site is blocked by the last helix in the DH domain and the two PH domains. This conformation is stabilized by multiple interactions among the domains and two well-structured inter-domain linkers .

• Phosphorylation: FARP2 is phosphorylated by aPKCι at two conserved sites (S340 and S370) located in the FERM-FA domain. This phosphorylation affects FARP2's interaction with aPKCι but interestingly does not directly impact its GEF activity .

• Protein-protein interactions: FARP2 forms complexes with various proteins including aPKCι through a RIPR motif-dependent interaction. The binding occurs specifically between the FERM and FERM-adjacent (FA) domains of FARP2 and the kinase domain of aPKCι .

• Feedback mechanisms: FARP2 participates in a positive feedback loop with aPKCι, where FARP2 increases Cdc42-GTP levels, which activates aPKCι through PAR6, and activated aPKCι in turn regulates FARP2 localization and function through phosphorylation .

What are the validated applications for FARP2 antibodies?

Current commercial FARP2 antibodies have been validated for several research applications:

When selecting a FARP2 antibody for your research, consider the specific application requirements and the host species of your experimental model. Most commercially available antibodies are polyclonal and produced in rabbits .

What are the key considerations for optimizing FARP2 antibody use in immunohistochemistry?

For optimal results in FARP2 immunohistochemistry:

• Tissue preparation: Use proper fixation techniques (typically 10% neutral buffered formalin) and paraffin embedding. Ensure adequate tissue sectioning (4-6 μm thickness).

• Antigen retrieval: FARP2 antibodies often require heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0). Test both methods to determine optimal conditions.

• Antibody dilution: Start with the manufacturer's recommended dilution (typically 1:50-1:200 for IHC applications) and optimize as needed. Perform a dilution series to determine the optimal antibody concentration for your specific tissue.

• Blocking: Use appropriate blocking solutions to minimize background staining. A combination of serum (from the species of the secondary antibody) and protein blockers is often effective.

• Controls: Always include positive controls (tissues known to express FARP2, such as liver cancer tissue) and negative controls (primary antibody omitted or isotype control).

• Detection system: Select an appropriate detection system compatible with your primary antibody's host species. DAB (3,3'-diaminobenzidine) is commonly used for visualization.

• Counterstaining: Use hematoxylin for nuclear counterstaining to provide cellular context for FARP2 localization.

How can I investigate FARP2's role in the aPKCι-FARP2-Cdc42 feedback loop?

To study the aPKCι-FARP2-Cdc42 feedback loop effectively:

• Protein interaction analysis:

  • Perform co-immunoprecipitation (co-IP) experiments using FARP2 antibodies to pull down protein complexes and probe for aPKCι and other components.

  • Use FRET-FLIM (Fluorescence Resonance Energy Transfer-Fluorescence Lifetime Imaging Microscopy) to detect direct protein interactions in live cells .

  • Consider proximity ligation assays (PLA) to visualize protein-protein interactions in situ.

• Phosphorylation studies:

  • Examine FARP2 phosphorylation using phospho-specific antibodies targeting S340 and S370 .

  • Employ phosphorylation-resistant mutants (S340A/S370A) to analyze the functional consequences of FARP2 phosphorylation by aPKCι.

  • Use aPKCι inhibitors (e.g., CRT0066854) to assess phosphorylation-dependent effects .

• GEF activity assays:

  • Measure Cdc42 activation using G-LISA or PAK1-PBD pulldown assays after manipulating FARP2 expression or phosphorylation status .

  • Compare GEF activity between wild-type FARP2 and phosphorylation-resistant mutants.

• Localization studies:

  • Perform immunofluorescence using FARP2 antibodies to track subcellular localization changes in response to aPKCι activation or inhibition.

  • Use live-cell imaging with fluorescently tagged FARP2 constructs to monitor dynamic localization changes.

• Functional readouts:

  • Assess polarity and junction formation (e.g., trans-epithelial electrical resistance, localization of junctional proteins) after manipulating components of the feedback loop.

What approaches can resolve conflicting reports about FARP2's substrate specificity for Rac1 versus Cdc42?

The literature contains conflicting reports about whether FARP2 predominantly activates Rac1 or Cdc42 . To resolve this conflict:

• Comparative direct GEF activity measurements:

  • Perform in vitro GEF activity assays using purified components (FARP2, Rac1, and Cdc42) under identical conditions.

  • Use multiple established methodologies such as fluorescence-based nucleotide exchange assays and radiolabeled GTP binding assays.

• Cellular activation patterns:

  • Measure activation of both Rac1 and Cdc42 simultaneously in various cell types using G-LISA or pull-down assays after FARP2 manipulation.

  • Develop FRET-based biosensors specific for each GTPase to monitor spatiotemporal activation patterns in living cells.

• Domain-specific mutations:

  • Introduce mutations in the DH domain of FARP2 at residues that differ from typical DH domains (e.g., His690 instead of Lys/Arg, Gln727 instead of Asn) .

  • Assess how these mutations affect GEF activity toward Rac1 versus Cdc42.

• Context-dependent activation:

  • Examine whether FARP2's substrate preference varies depending on:

    • Cell type and expression patterns of scaffolding proteins

    • Activation state of upstream signaling molecules

    • Post-translational modifications of FARP2

    • Subcellular localization

• Structural analysis:

  • Use structural biology approaches (X-ray crystallography, cryo-EM) to visualize FARP2 interactions with both Rac1 and Cdc42.

  • Compare these structures to identify determinants of specificity.

What validation steps should be performed when using a new FARP2 antibody for research?

When introducing a new FARP2 antibody into your research workflow:

• Specificity validation:

  • Perform western blot analysis to confirm the antibody detects a protein of the expected molecular weight (~120 kDa) .

  • Include positive controls (tissues/cells known to express FARP2) and negative controls (FARP2 knockdown cells using siRNA or CRISPR).

  • Consider testing multiple antibodies targeting different epitopes to confirm specificity.

• Cross-reactivity assessment:

  • Test the antibody in samples from multiple species if cross-species reactivity is claimed.

  • Evaluate potential cross-reactivity with the closely related FARP1 protein, which shares high sequence homology (~60%) .

• Application-specific validation:

  • For each intended application (WB, IHC, IF, ELISA), perform specific validation experiments.

  • Optimize critical parameters (antibody concentration, incubation time, buffer composition) for each application.

• Reproducibility assessment:

  • Test the antibody across multiple batches of the same sample to ensure consistent results.

  • Document all experimental conditions thoroughly to ensure reproducibility.

• Functional validation:

  • Correlate antibody staining/detection with functional readouts of FARP2 activity.

  • Confirm that experimentally induced changes in FARP2 expression are accurately reflected in antibody detection.

How can phosphorylation-specific FARP2 antibodies be developed and validated?

Developing phosphorylation-specific antibodies for FARP2 S340 and S370 sites is valuable for studying aPKCι-dependent regulation . The process involves:

• Peptide design:

  • Design phosphopeptides containing the target phosphorylation sites (S340 and S370) with surrounding amino acid sequences.

  • Include both phosphorylated and non-phosphorylated versions of each peptide.

• Antibody production:

  • Immunize rabbits or other suitable hosts with the phosphopeptides conjugated to carrier proteins.

  • Perform ELISA screening to identify antibodies with high affinity and specificity.

  • Purify antibodies using phosphopeptide affinity chromatography.

• Specificity validation:

  • Test antibodies against phosphorylated and non-phosphorylated peptides.

  • Perform dot blots or western blots with recombinant FARP2 proteins (wild-type and S340A/S370A mutants) phosphorylated in vitro by aPKCι.

  • Validate in cellular contexts by comparing:

    • Cells expressing wild-type FARP2 versus phosphorylation-site mutants

    • Cells treated with or without aPKCι inhibitors (e.g., CRT0066854)

    • Cells with or without aPKCι knockdown/knockout

• Functional validation:

  • Correlate phospho-antibody signals with functional outcomes of aPKCι-FARP2 signaling.

  • Use phospho-antibodies to track changes in FARP2 phosphorylation during biological processes such as junction formation or polarity establishment.

• Application optimization:

  • Optimize conditions for each application (western blotting, immunoprecipitation, immunofluorescence).

  • Determine requirements for phosphatase inhibitors during sample preparation.

What are common issues when detecting FARP2 in western blots and how can they be resolved?

When working with FARP2 antibodies in western blotting:

Additional optimization strategies:

• For FARP2 detection, use TBST (Tris-buffered saline with 0.1% Tween-20) rather than PBST for washes to reduce background.

• Consider using gradient gels (4-15%) to better resolve high molecular weight proteins.

• Include phosphatase inhibitors if studying phosphorylated forms of FARP2.

• Store antibodies according to manufacturer recommendations (typically at -20°C with glycerol) .

How can subcellular localization of FARP2 be accurately determined using immunofluorescence?

For precise subcellular localization of FARP2:

• Sample preparation optimization:

  • Test different fixation methods (paraformaldehyde, methanol, or glutaraldehyde) to determine which best preserves FARP2 epitopes while maintaining cellular architecture.

  • Optimize permeabilization conditions to ensure antibody access to intracellular FARP2 while preserving membrane structures.

• Antibody validation for IF:

  • Verify specificity using FARP2 knockdown or knockout cells as negative controls.

  • Confirm localization patterns with multiple antibodies targeting different FARP2 epitopes.

  • Use tagged FARP2 constructs (e.g., GFP-FARP2) as complementary approaches.

• Co-localization studies:

  • Perform double immunofluorescence with markers for:

    • Cell junctions (E-cadherin, ZO-1) to assess FARP2's role in junction formation

    • Polarity complexes (aPKCι, PAR6) to investigate the aPKCι-FARP2 interaction

    • Active Cdc42 to examine the relationship between FARP2 and its substrate

  • Use appropriate statistical measures (Pearson's correlation coefficient, Manders' overlap coefficient) to quantify co-localization.

• Super-resolution microscopy:

  • Employ techniques like STED, STORM, or PALM to resolve FARP2 localization beyond the diffraction limit.

  • This is particularly important for studying FARP2 at cell junctions or other complex subcellular structures.

• Live-cell imaging:

  • Use fluorescently tagged FARP2 constructs for dynamic localization studies.

  • Apply FRAP (Fluorescence Recovery After Photobleaching) to assess FARP2 mobility and turnover at specific subcellular locations.

• Stimulus-dependent relocalization:

  • Track FARP2 localization changes in response to:

    • aPKCι activation or inhibition

    • Junction formation or disruption

    • Plexin signaling activation

How can FARP2 antibodies be applied to study its role in neurological disorders?

FARP2 has been implicated in neurological disorders including autism and schizophrenia . To investigate these connections:

• Expression analysis in patient samples:

  • Use FARP2 antibodies for IHC or western blot analysis of post-mortem brain tissue from patients with neurological disorders versus controls.

  • Examine FARP2 expression patterns across different brain regions and cell types using single-cell approaches.

• Functional studies in neuronal cultures:

  • Employ FARP2 antibodies for immunofluorescence to track subcellular localization in primary neuronal cultures.

  • Investigate co-localization with synaptic markers to assess FARP2's role in synaptic plasticity.

  • Correlate FARP2 expression/localization with neuronal morphology and connectivity.

• Animal model validation:

  • Use FARP2 antibodies to characterize expression patterns in animal models of neurodevelopmental disorders.

  • Correlate FARP2 dysregulation with behavioral phenotypes and neuroanatomical abnormalities.

• Mechanism investigation:

  • Study FARP2's interaction with plexins and semaphorins, which are involved in axon guidance and have been implicated in neurological disorders .

  • Examine how disease-associated mutations affect FARP2 expression, localization, and function using mutation-specific cellular models.

• Therapeutic target assessment:

  • Use FARP2 antibodies to monitor protein expression and pathway activation in response to potential therapeutic interventions.

  • Develop cell-based assays incorporating FARP2 antibodies for high-throughput screening of compounds that modulate FARP2-dependent signaling.

What role does FARP2 play in the auto-inhibition mechanism and how can this be studied?

The crystal structure of FARP2 reveals an auto-inhibited conformation that regulates its GEF activity . To investigate this mechanism:

• Structure-function analysis:

  • Generate targeted mutations that disrupt the auto-inhibitory interactions between the DH domain, PH domains, and inter-domain linkers.

  • Create truncation constructs that remove specific auto-inhibitory elements.

  • Measure GEF activity of these mutants compared to wild-type FARP2 using in vitro and cellular assays.

• Regulatory mechanisms:

  • Investigate whether post-translational modifications (particularly aPKCι-mediated phosphorylation at S340 and S370) affect the auto-inhibited conformation .

  • Examine if protein-protein interactions (e.g., with aPKCι or plexins) induce conformational changes that relieve auto-inhibition.

• Structural approaches:

  • Use hydrogen-deuterium exchange mass spectrometry (HDX-MS) to detect conformational changes in FARP2 under different conditions.

  • Apply single-molecule FRET to monitor conformational dynamics of FARP2 in solution.

  • Perform additional crystallographic studies of FARP2 in active conformations or bound to interaction partners.

• Cellular assays:

  • Develop FRET-based biosensors to monitor FARP2 conformational changes in living cells.

  • Correlate conformational state with subcellular localization and GEF activity.

  • Investigate whether different cellular contexts favor active or inactive FARP2 conformations.

• Therapeutic implications:

  • Explore the possibility of developing small molecules that specifically target the auto-inhibited conformation.

  • Assess whether disrupting auto-inhibition could be beneficial in conditions where increased FARP2 activity might be therapeutic.