ARHGAP18 Antibody

What is ARHGAP18 and what cellular functions does it regulate?

ARHGAP18 is a Rho GTPase-activating protein that specifically accelerates the hydrolysis of GTP of RhoA in epithelial cells, though it also exhibits increased activity toward RhoC in endothelial cells . This protein functions as a crucial regulator of:

• Cell shape, migration, and spreading through RhoA inactivation

• Actin cytoskeleton organization, particularly in microvilli formation

• Formation of an autoregulatory module with ezrin to fine-tune local levels of active RhoA

• Endothelial cell alignment in response to high shear laminar flow

• Cell motility during wound healing and in Boyden chamber migration assays

ARHGAP18 contains a C-terminal RhoGAP domain without other specific domains and is expressed in most organs, showing diffuse cytoplasmic localization with enrichment at the leading edge during cell migration and at membrane protrusions during cell spreading .

What types of ARHGAP18 antibodies are commercially available for research?

Several types of ARHGAP18 antibodies are available for research applications:

Most commercially available antibodies are rabbit polyclonal antibodies targeting different epitopes of the ARHGAP18 protein .

How should I optimize Western blot protocols for ARHGAP18 detection?

For optimal Western blot detection of ARHGAP18:

• Sample preparation: ARHGAP18 typically appears as two immunoreactive bands around 90 kDa, both of which represent ARHGAP18 (the slower-migrating form is likely due to phosphorylation) .

• Antibody dilution: Use dilution ranges of 1:500-1:3000 depending on the specific antibody. For example, Proteintech's 28409-1-AP antibody is recommended at 1:500-1:3000 for Western blot .

• Controls: Include both positive controls (HeLa cells, MDA-MB-453s cells) and negative controls (ARHGAP18-knockdown cells) .

• Blocking: Use PBS containing 0.5% BSA to reduce background while maintaining specific signal .

• Detection method: HRP-conjugated secondary antibodies or directly HRP-conjugated primary antibodies (like NBP2-81704H) can be used depending on sensitivity requirements .

Studies have shown that immunoblotting can detect both endogenous ARHGAP18 and overexpressed tagged versions, with expression particularly high in most organs except small intestine .

What are the critical parameters for successful immunofluorescence detection of ARHGAP18?

For optimal immunofluorescence detection of ARHGAP18:

• Fixation: 4% paraformaldehyde for 15 minutes maintains both cellular morphology and ARHGAP18 epitopes.

• Permeabilization: 0.1% Triton X-100 is recommended for accessing intracellular ARHGAP18.

• Dilution: Typically 1:50-1:500, with Proteintech's antibody recommended at 1:50-1:500 .

• Subcellular localization patterns to expect:

  • Diffuse cytoplasmic localization in most cells

  • Enrichment at the leading edge of migrating cells

  • Localization to membrane protrusions during cell spreading

  • Enrichment at the apical surface and microvilli in polarized epithelial cells

  • Nuclear localization in some cellular contexts

  • Localization to angiogenic endothelial cell junctions

• Co-staining: Combine with ezrin antibodies to study the ARHGAP18-ezrin interaction at microvilli .

When evaluating staining specificity, compare with ARHGAP18 siRNA-transfected cells as a negative control .

How can ARHGAP18 antibodies be utilized to investigate its role in cytoskeletal reorganization?

ARHGAP18 antibodies can be leveraged to study cytoskeletal reorganization through several sophisticated approaches:

• Co-immunoprecipitation assays: ARHGAP18 antibodies can be used to pull down protein complexes to identify interaction partners. This approach revealed that ARHGAP18 co-precipitates with flag-ezrin and that truncated ezrin 1-479 bound at approximately tenfold the level compared to full-length protein .

• Super-resolution microscopy: When combined with STORM (Stochastic Optical Reconstruction Microscopy), ARHGAP18 antibodies help visualize actin filament networks at <40nm resolution, revealing that loss of ARHGAP18 results in near-total loss of basal actin bundles, including stress fibers and filopodia .

• Proximity ligation assays: These can detect the direct interaction between ARHGAP18 and ezrin at the subcellular level, helping investigate how this interaction regulates RhoA activity in microvilli .

• Live cell imaging: Combining immunofluorescence with time-lapse imaging after ARHGAP18 knockdown demonstrated that cells exhibited active membrane blebs outward and gradually spread to take a stable morphology, revealing ARHGAP18's role in proper membrane protrusion formation .

• Transmission electron microscopy (TEM): While not directly using antibodies, TEM analysis of ARHGAP18-knockout cells revealed that actin filaments within microvilli lacked distinct parallel aligned actin core bundles found in wildtype cells .

What approaches can determine if ARHGAP18 is properly targeted in my experimental system?

To confirm proper ARHGAP18 targeting:

• Western blot validation: Two immunoreactive bands around 90 kDa should be detected with ARHGAP18 antibodies. Both bands should disappear after ARHGAP18 siRNA treatment, confirming specificity .

• Subcellular localization analysis:

  • In wildtype cells: Diffuse cytoplasmic staining with enrichment at specific cellular structures depending on context (microvilli, leading edge, etc.)

  • In ARHGAP18-knockout/knockdown cells: Loss of specific cytoplasmic staining while nonspecific nuclear staining remains

• Functional readouts:

  • Cell morphology changes: Increased rounded cells after ARHGAP18 knockdown

  • RhoA activity: Enhanced RhoA-GTP levels at the apical surface detected with AHPH-GFP biosensor in ARHGAP18-knockout cells

  • Microvilli alterations: Increased number but shortened microvilli in ARHGAP18-knockout cells

  • Cell migration: Delayed wound closure in ARHGAP18 siRNA-treated cells

• Rescue experiments: Expression of full-length tagged ARHGAP18 should rescue phenotypes in knockout cells, while expression of just the GAP domain or catalytically inactive mutants (ARHGAP18(R356A)) may not fully rescue, confirming the importance of both proper targeting and GAP activity .

How do I analyze ARHGAP18's interaction with ezrin and its implications for microvilli regulation?

Analysis of the ARHGAP18-ezrin interaction requires multiple complementary approaches:

• Biochemical interaction analysis:

  • Co-immunoprecipitation assays have shown that endogenous ARHGAP18 co-precipitates with flag-ezrin

  • Truncated ezrin 1-479 lacking the C-terminal regulatory domain bound ARHGAP18 at approximately tenfold the level compared to full-length protein, suggesting that active "open" ezrin preferentially interacts with ARHGAP18

• Structural predictions:

  • AlphaFold2 has been used to predict the structure and protein-protein interactions between full-length human ARHGAP18 and human ezrin FERM domain

• Functional analysis of microvilli regulation:

  • ARHGAP18 knockout cells exhibit both increased microvilli number and enhanced RhoA activity at the apical surface

  • This occurs through two simultaneous mechanisms:
    a) Enhanced RhoA activity increases ezrin phosphorylation via LOK/SLK pathway
    b) Increased RhoA activation leads to aberrant non-muscle myosin-2 activity inside microvilli

• Rescue experiments with domain mutants:

  • Expression of full-length ARHGAP18 rescues the phenotype

  • Expression of just the GAP domain shows diffuse localization and fails to rescue

  • Expression of catalytically inactive ARHGAP18(R356A) localizes correctly but doesn't rescue, indicating both proper targeting and GAP activity are essential

The data collectively demonstrates that ARHGAP18-ezrin functions as an autoregulatory module where ezrin recruits ARHGAP18 to microvilli, which then locally reduces RhoA activity to maintain proper microvilli length and organization .

What controls should I include when studying ARHGAP18's role in RhoA regulation and cytoskeletal organization?

A comprehensive set of controls is essential for studying ARHGAP18's role in RhoA regulation:

• Genetic controls:

  • Positive control: Wildtype cells showing normal ARHGAP18 expression and function

  • Negative control: ARHGAP18 knockout/knockdown cells

  • Rescue controls:

    • Full-length ARHGAP18 (should fully rescue)

    • GAP domain only (should not fully rescue)

    • Catalytically inactive R356A mutant (should not rescue GAP-dependent functions)

• Activity assays:

  • RhoA-GTP pulldown assay: Using GST-Rhotekin-RBD to precipitate active GTP-bound RhoA

  • RhoA biosensor: AHPH-GFP biosensor that preferentially binds active RhoA-GTP to visualize local RhoA activity

• Downstream pathway controls:

  • ROCK pathway: Measure phosphorylation of myosin light chain (pMLC)

  • LIMK pathway: Measure phosphorylation of LIMK (pLIMK) and cofilin (pCofilin)

  • LOK/SLK pathway: Measure phosphorylation of ezrin/ERM proteins (pERM)

• Functional readouts:

  • Cell morphology: Roundedness, ability to form protrusions

  • Cell migration: Wound healing, Boyden chamber assays

  • Actin organization: Stress fiber formation, microvilli structure

  • Ultrastructural analysis: TEM to visualize individual actin filaments in microvilli

• Drug controls:

  • ROCK inhibitor (Y-27632) to test ROCK dependency

  • Blebbistatin (myosin II inhibitor) to test myosin II dependency

These controls allow for comprehensive analysis of ARHGAP18's role in regulating RhoA and downstream cytoskeletal organization.

How can ARHGAP18 antibodies be utilized in cancer research and prognostic studies?

ARHGAP18 antibodies have significant applications in cancer research:

• Expression pattern analysis:

  • IHC analysis of ARHGAP18 in breast cancer tissue revealed that ARHGAP18 protein is expressed in both the cytoplasm and nuclei of tumor cells

  • Loss of cytoplasmic expression shows associations with lymphovascular invasion (LVI)

  • Loss of nuclear expression is associated with higher grade, HER2+ status, and high Ki67LI

• Prognostic marker validation:

  • Breast cancer studies have shown that cytoplasmic and nuclear ARHGAP18 expression is positively associated with improved survival independent of other variables (P=0.01, HR=0.74, 95% CI 0.60–87)

  • Transcriptomic profiling identified ARHGAP18 as a gene associated with lymphovascular invasion in breast cancer

• Mechanism investigation in tumor progression:

  • ARHGAP18 antibodies can help investigate its role in epithelial-mesenchymal transition, as loss of cytoplasmic expression shows associations with EMT in breast cancer

  • The interaction between ARHGAP18 and Hippo pathway components (like YAP) can be examined in cancer contexts using co-immunoprecipitation and co-localization studies

• Therapeutic target assessment:

  • As ARHGAP18 expression is associated with better outcomes, antibodies can help assess its potential as a therapeutic target or biomarker

  • Understanding mechanistic roles through antibody-based techniques can identify pathway vulnerabilities for therapeutic intervention

What methodological approaches should be used to study ARHGAP18's role in endothelial cell function and angiogenesis?

Studying ARHGAP18 in endothelial contexts requires specialized approaches:

• Localization studies in endothelial cells:

  • ARHGAP18 is localized to angiogenic endothelial cell junctions

  • In flow-adapted endothelial cells, ARHGAP18 plays a role in alignment response

• Functional assays for angiogenesis:

  • Wound healing assays: ARHGAP18 knockdown results in irregular and protruded migratory front with disrupted cell-cell junctions at the leading edge

  • 3D spheroid sprouting assay: Knockdown of ARHGAP18 results in significant increases in both the number of sprouts and the cumulative sprout length of the spheroids

  • Flow-induced alignment: ARHGAP18 depletion inhibits the alignment of ECs in the direction of flow and promotes inflammatory phenotypes

• Molecular pathway analysis:

  • Examine effects on inflammatory markers (NF-κB, ICAM-1) and eNOS in endothelial cells

  • In atherosclerosis models, examine EC alignment and inflammatory response in ARHGAP18-deficient mice

• In vivo models:

  • Apolipoprotein E−/−Arhgap18−/− double-mutant mice on high-fat diets show early onset of atherosclerosis with lesions developing in atheroprotective regions

  • These approaches collectively demonstrate ARHGAP18's role as a protective gene that maintains EC alignment in response to flow

What are common technical issues when using ARHGAP18 antibodies and how can they be resolved?

How can I determine the optimal ARHGAP18 antibody for my specific research application?

Selection of the optimal ARHGAP18 antibody depends on several factors:

• Consider the target region and epitope:

  • Internal region antibodies (ABIN6257900) for general detection

  • C-terminal antibodies for specific domain targeting

  • AA 517-566 region antibodies for specific epitope recognition

• Match species reactivity to your model system:

  • Human-only reactivity: Some antibodies only recognize human ARHGAP18

  • Multi-species reactivity: Some antibodies recognize human, rat, and mouse ARHGAP18

• Validate application compatibility:

  • Western blot: Most antibodies are validated for this application

  • Immunofluorescence: Check for specific validation in IF/ICC

  • Flow cytometry: Only certain antibodies are validated for FACS

  • Immunoprecipitation: Limited antibodies are validated for IP

• Experimental validation approaches:

  • Test antibody on positive control samples (HeLa cells, MDA-MB-453s cells)

  • Compare with negative controls (ARHGAP18 knockout/knockdown cells)

  • Confirm expected molecular weight (approximately 90 kDa with two bands)

  • Verify expected subcellular localization patterns (cytoplasmic, not nuclear)

• Consider conjugation requirements:

  • Unconjugated for flexible detection methods

  • HRP-conjugated for direct detection in Western blot

  • Fluorophore-conjugated for direct detection in IF/FACS