ARHGAP5 Antibody

What is ARHGAP5 and why is it important in cell signaling research?

ARHGAP5, also known as p190-B or RHOGAP5, is a 172.46 kDa Rho GTPase-activating protein located in human chromosome 14q12 (14; NC_000014.9 (32072588..32159728)) . It functions as a GTPase-activating protein for Rho family members and plays a critical role in regulating cytoskeletal dynamics and cell signaling pathways . The protein is primarily detected in skin fibroblasts at the protein level and is distributed in the cytoplasm and cell membrane as a peripheral membrane protein .

ARHGAP5 is particularly important in research because it colocalizes with the alpha5-beta1 integrin receptor (ITGA5/ITGB1) for fibronectin in a fibrillar pattern , suggesting its involvement in cell-matrix interactions. Additionally, it may play a role in reducing the p21rasGTPase-activating potential of p120GAP, indicating its importance in multiple signaling networks .

Which cellular models are most appropriate for ARHGAP5 expression studies?

Based on experimental validation data, multiple cell lines have been confirmed to express ARHGAP5 and can serve as positive controls for antibody validation:

When designing experiments to study ARHGAP5, these cell lines provide reliable expression systems for initial validation and characterization. For comparative studies, it would be advisable to include both high-expressing and low-expressing lines to establish a dynamic range for your detection system.

What are the recommended protocols for Western blot analysis using ARHGAP5 antibodies?

For optimal Western blot results with ARHGAP5 antibodies, follow these methodological guidelines:

• Sample preparation: Extract proteins using standard lysis buffers containing protease inhibitors to prevent degradation of the 172-190 kDa ARHGAP5 protein.

• Antibody selection and dilution: Multiple validated antibodies are available with specific recommended dilutions:

  • Proteintech antibody 55165-1-AP: Use at 1:500-1:1000 dilution

  • Boster Bio antibody A05701: Use at 1:500-1:2000 dilution

  • Abbexa antibody: Use at 1:500-1:2000 dilution

• Gel and transfer conditions: Use low percentage (6-8%) SDS-PAGE gels to adequately resolve the high molecular weight ARHGAP5 protein (observed MW: 170-190 kDa) .

• Detection and visualization: Based on the antibody specifications, the ARHGAP5 protein should be detected at approximately 170-190 kDa. Variations in observed molecular weight (170-190 kDa) may result from post-translational modifications or differences in gel running conditions .

• Controls: Include validated positive controls such as lysates from HeLa, Jurkat, HepG2, or K-562 cells, which have been confirmed to express ARHGAP5 .

It is recommended to optimize these conditions for your specific experimental system to ensure reproducible results.

How can ARHGAP5 antibodies be used to study its phosphorylation status and signaling mechanisms?

ARHGAP5 undergoes important post-translational modifications that regulate its activity. Specifically, it can be directly phosphorylated on a single identified tyrosine residue by activated insulin (INS) and insulin-like growth factor-1 (IGF-1) receptors . Additionally, it has been shown to be tyrosine phosphorylated by cSrc and vSrc kinases .

To study these phosphorylation events and their impact on signaling:

• Phosphorylation-specific detection approach:

  • Use general ARHGAP5 antibodies to immunoprecipitate the protein from lysates of cells treated with or without growth factors/kinase activators

  • Perform Western blot with anti-phosphotyrosine antibodies to detect phosphorylation changes

  • Alternatively, use phospho-specific antibodies if available for the specific modification sites

• Functional analysis methodology:

  • Correlate phosphorylation status with ARHGAP5 GAP activity using in vitro GAP assays

  • Examine how phosphorylation affects ARHGAP5's ability to attenuate the signal transducing activity of Rac, Rho, and CDC42 GTPases

  • Investigate the relationship between phosphorylation state and interaction with the alpha5-beta1 integrin receptor

• Signal pathway integration:

  • Design experiments to determine how ARHGAP5 phosphorylation influences its role in reducing the p21rasGTPase-activating potential of p120GAP

  • Analyze crosstalk between Rho and Ras pathways through phosphorylation-dependent regulation of ARHGAP5

This multi-faceted approach allows researchers to connect phosphorylation events to specific functional outcomes in ARHGAP5-mediated signaling networks.

What methodological approaches are recommended for studying ARHGAP5 interactions with integrin receptors?

ARHGAP5 has been shown to colocalize with the alpha5-beta1 integrin receptor (ITGA5/ITGB1) for fibronectin in a fibrillar pattern . To study these interactions:

• Immunofluorescence co-localization protocol:

  • Fixed cell preparation: Fix cells with 4% paraformaldehyde, permeabilize with 0.1% Triton X-100

  • Primary antibody incubation: Use ARHGAP5 antibody (Boster Bio A05701 at 1:50-1:200 dilution for ICC/IF) together with alpha5-beta1 integrin antibodies

  • Secondary antibody selection: Use spectrally distinct fluorophore-conjugated secondary antibodies

  • Analysis: Perform confocal microscopy and calculate Pearson's correlation coefficient for co-localization quantification

• Biochemical interaction analysis:

  • Co-immunoprecipitation: Immunoprecipitate with anti-ARHGAP5 antibodies and probe for alpha5-beta1 integrin, or vice versa

  • Proximity ligation assay: Detect protein-protein interactions in situ with higher sensitivity than conventional co-localization

  • FRET/BRET analysis: For studying dynamic interactions in living cells

• Functional relationship assessment:

  • Integrin clustering induction: Based on findings that Rho proteins cluster after integrin cross-linking (PubMed:8537347)

  • Cell adhesion assays: Determine if ARHGAP5 knockdown or overexpression affects integrin-mediated adhesion to fibronectin

  • Migration assays: Assess the role of ARHGAP5-integrin interactions in directional cell movement

These methodological approaches provide complementary data about the spatial, biochemical, and functional aspects of ARHGAP5-integrin interactions.

How can researchers distinguish between closely related ARHGAP family members in experimental systems?

Distinguishing between similar ARHGAP family members requires careful experimental design:

• Antibody validation strategy:

  • Cross-reactivity testing: Test ARHGAP5 antibodies against recombinant proteins of related ARHGAP family members

  • Immunodepletion experiments: Perform sequential immunoprecipitations to separate specific ARHGAP proteins

  • Knockout/knockdown validation: Use siRNA or CRISPR-edited cells lacking ARHGAP5 to confirm antibody specificity

• Detection optimization:

  • Western blot conditions: Utilize the molecular weight differences among ARHGAP family members (ARHGAP5 observed at 170-190 kDa)

  • Epitope mapping: Select antibodies targeting unique regions not conserved among family members

  • Isoform-specific detection: Design PCR primers or choose antibodies that discriminate between splice variants

• Functional discrimination techniques:

  • Domain-specific activity assays: Target the GAP domain of ARHGAP5 that attenuates signal transduction of Rac, Rho, and CDC42

  • Protein interaction profiles: Identify specific binding partners unique to ARHGAP5, such as its association with alpha5-beta1 integrin

  • Subcellular localization patterns: Use high-resolution microscopy to distinguish localization differences between family members

These approaches help researchers ensure their experiments specifically measure ARHGAP5 without interference from related proteins, increasing the reliability of their findings.

What are the critical controls needed for ARHGAP5 antibody validation in immunological techniques?

Proper validation of ARHGAP5 antibodies requires rigorous controls:

• Positive and negative cellular controls:

  • Positive controls: Use cell lines with confirmed ARHGAP5 expression, such as HeLa, Jurkat, HepG2, and K-562 cells

  • Negative controls: Implement ARHGAP5 knockdown/knockout cells or tissues known not to express the protein

  • Expression gradient: Include samples with varying expression levels to assess dynamic range of detection

• Antibody-specific controls:

  • Blocking peptide competition: Pre-incubate antibody with immunizing peptide to confirm specificity (blocking peptides are available for purchase from manufacturers)

  • Secondary antibody only: Omit primary antibody to identify non-specific binding of secondary antibodies

  • Isotype control: Use matched isotype IgG at the same concentration as the primary antibody

• Technical validation approaches:

  • Multiple antibody concordance: Compare results using different antibodies targeting distinct epitopes of ARHGAP5

  • Multiple technique validation: Confirm findings across Western blot, immunofluorescence, and ELISA platforms

  • Recombinant protein standards: Include purified ARHGAP5 protein as size/specificity reference

Following these methodological controls ensures that experimental observations are specifically attributed to ARHGAP5 rather than artifacts or cross-reactive signals.

What approaches can resolve discrepancies in observed molecular weights of ARHGAP5 in different experimental systems?

Researchers often observe variations in ARHGAP5 molecular weight across different studies and systems:

To resolve these discrepancies:

• Technical optimization:

  • Standardize gel percentage, running buffer, and electrophoresis conditions

  • Use gradient gels to better resolve high molecular weight proteins

  • Include molecular weight markers spanning the range of interest (150-200 kDa)

• Biological explanation investigation:

  • Analyze post-translational modifications: Phosphorylation by INS/IGF-1 receptors or cSrc/vSrc may alter apparent molecular weight

  • Identify tissue/cell-specific isoforms: Compare full-length vs. truncated forms across cell types

  • Deglycosylation assays: Treat samples with glycosidases to identify contribution of glycosylation to apparent size

• Methodological resolution approaches:

  • Mass spectrometry analysis: Precisely determine the actual molecular weight and modifications

  • 2D gel electrophoresis: Separate proteins by both isoelectric point and molecular weight to identify variants

  • Western blot with epitope-mapped antibodies: Determine if size variations correlate with specific protein domains

Understanding these variations is crucial for correctly interpreting experimental data and identifying possible functional modifications of ARHGAP5.

How can researchers optimize co-immunoprecipitation protocols to study ARHGAP5 protein-protein interactions?

Co-immunoprecipitation (Co-IP) is valuable for studying ARHGAP5 interactions, particularly with proteins like alpha5-beta1 integrin receptor and components of Rho/Ras signaling pathways :

• Lysis buffer optimization:

  • Mild detergent selection: Use digitonin (0.5-1%) or CHAPS (0.5-1%) for membrane protein interactions

  • Salt concentration: Start with physiological levels (150mM NaCl) and adjust based on interaction strength

  • Phosphatase inhibitors: Include sodium orthovanadate and sodium fluoride to preserve phosphorylation-dependent interactions, particularly important for ARHGAP5 studies

• Antibody selection criteria:

  • Choose antibodies validated for immunoprecipitation applications

  • Test multiple antibodies targeting different ARHGAP5 epitopes

  • Consider whether N-terminal or C-terminal antibodies might interfere with specific protein interactions

• Technical protocol refinements:

  • Pre-clearing strategy: Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Cross-linking option: Consider reversible cross-linking for transient interactions

  • Elution methods: Use either denaturing (SDS) or non-denaturing (competing peptide) elution depending on downstream applications

• Controls and validation:

  • Reverse Co-IP: Immunoprecipitate with antibodies against the interacting partner and blot for ARHGAP5

  • IgG control: Use matched isotype IgG as negative control

  • Input control: Include 5-10% of pre-IP lysate to verify expression levels

  • Validation with orthogonal methods: Confirm interactions using proximity ligation assay or FRET

These methodological refinements maximize the chances of detecting genuine ARHGAP5 interactions while minimizing artifacts and false positives.

How can ARHGAP5 antibodies be applied in studying mechanotransduction and cytoskeletal dynamics?

ARHGAP5's role as a Rho GTPase-activating protein and its association with integrin receptors position it as a key player in mechanotransduction pathways:

• Live-cell imaging applications:

  • ARHGAP5-GFP fusion protein localization during cell migration and adhesion

  • Correlation of ARHGAP5 dynamics with Rho GTPase activity using FRET-based biosensors

  • Super-resolution microscopy to visualize ARHGAP5 clustering with integrins following mechanical stimulation

• Cytoskeletal reorganization analysis:

  • Quantification of stress fiber formation, focal adhesion dynamics, and lamellipodia extension in cells with modulated ARHGAP5 levels

  • Correlation of ARHGAP5 phosphorylation status with cytoskeletal changes following mechanical stress

  • Analysis of how ARHGAP5 GAP activity toward Rac, Rho, and CDC42 differentially regulates distinct cytoskeletal structures

• Mechanotransduction pathway mapping:

  • Determination of how integrin clustering after cross-linking affects ARHGAP5 activity and localization

  • Investigation of ARHGAP5's role in translating matrix stiffness sensing to intracellular signaling cascades

  • Analysis of how mechanical forces influence ARHGAP5 phosphorylation by INS/IGF-1 receptors or cSrc/vSrc

These approaches can reveal how ARHGAP5 functions as a mechanosensitive regulator of cell behavior through its effects on the cytoskeleton and signaling pathways.

What methodological approaches can assess the role of ARHGAP5 in cross-talk between Rho and Ras signaling pathways?

ARHGAP5 may serve as an integration point between Rho and Ras signaling, as it potentially reduces the p21rasGTPase-activating potential of p120GAP :

• Pathway crosstalk experimental design:

  • Simultaneous measurement of Rho and Ras activity using pathway-specific biosensors in cells with ARHGAP5 manipulation

  • Analysis of how ARHGAP5's GAP domain activity toward Rho family GTPases affects Ras pathway activation

  • Determination of whether phosphorylation of ARHGAP5 by upstream kinases differentially regulates its effects on Rho versus Ras pathways

• Quantitative signaling analysis:

  • Phosphoproteomics to identify downstream targets of both pathways following ARHGAP5 modulation

  • Kinetic analysis of pathway activation/deactivation to detect temporal relationships and feedback mechanisms

  • Single-cell analysis to capture heterogeneity in pathway crosstalk that might be masked in population measurements

• Domain-specific functional studies:

  • Structure-function analysis using domain-specific mutations to separate ARHGAP5's effects on Rho versus Ras signaling

  • Identification of specific binding partners mediating pathway integration

  • Computational modeling of pathway interaction networks with ARHGAP5 as a node connecting Rho and Ras signaling

These methodological approaches can uncover how ARHGAP5 serves as a molecular switch coordinating distinct but interconnected signaling pathways in cellular responses.

How might emerging antibody technologies advance ARHGAP5 research beyond current applications?

Emerging technologies offer exciting possibilities for advancing ARHGAP5 research:

• Proximity labeling applications:

  • APEX2 or BioID fusion to ARHGAP5 to identify the complete interactome in living cells

  • Spatial-specific interactome mapping at different subcellular locations (membrane, cytoplasm, focal adhesions)

  • Stimulus-dependent interactome changes following growth factor treatment or mechanical stimulation

• Single-molecule analysis techniques:

  • Single-molecule pull-down (SiMPull) to determine stoichiometry of ARHGAP5 complexes

  • Super-resolution microscopy with antibody-based detection to visualize nanoscale organization

  • Single-molecule tracking to determine ARHGAP5 diffusion coefficients and confinement at specific cellular structures

• Integrated multi-omics approaches:

  • Correlation of ARHGAP5 phosphorylation status with global phosphoproteomic changes

  • Analysis of ARHGAP5 interactome in relation to transcriptomic changes following pathway modulation

  • Systems biology integration of proteomic, genomic, and metabolomic data to place ARHGAP5 in broader cellular networks

• Therapeutic targeting possibilities:

  • Development of conformation-specific antibodies to distinguish active versus inactive ARHGAP5

  • Intrabody applications to modulate ARHGAP5 function in specific subcellular compartments

  • Nanobody development for super-resolution imaging and acute functional perturbations

These emerging technological applications could transform our understanding of ARHGAP5's dynamic roles in cell signaling, cytoskeletal regulation, and disease processes.

What are the most reliable methodological approaches for quantifying ARHGAP5 expression and activity in experimental systems?

Based on the available evidence, researchers should consider these validated approaches:

• Expression quantification:

  • Western blot using antibodies from Proteintech (55165-1-AP, 1:500-1:1000) , Boster Bio (A05701, 1:500-1:2000) , or Abbexa (1:500-1:2000)

  • qRT-PCR with validated primers targeting conserved regions of the ARHGAP5 transcript

  • Immunofluorescence using antibodies validated for ICC/IF applications (Boster Bio A05701, 1:50-1:200)

• Activity measurement:

  • In vitro GAP assays measuring GTPase activity toward Rho family members

  • Phosphorylation status analysis as a proxy for activation state

  • Downstream effector monitoring (stress fiber formation, focal adhesion dynamics)

• Interaction profiling:

  • Co-immunoprecipitation to detect interaction with known partners including alpha5-beta1 integrin receptor

  • Proximity ligation assays for in situ detection of protein-protein interactions

  • FRET-based biosensors to monitor real-time activity in living cells