Phospho-ARHGAP35 (Tyr1105) Antibody

What is ARHGAP35 and what is the significance of Tyr1105 phosphorylation?

ARHGAP35 (also known as p190RhoGAP, p190A, or GRF-1) is a Rho GTPase-activating protein that inactivates RhoA by stimulating its intrinsic GTPase activity. Tyr1105 phosphorylation is a critical regulatory mechanism that mediates the direct interaction between ARHGAP35 and p120RasGAP through the latter's N-terminal SH2 domain .

This phosphorylation is mediated by several kinases, including Src family kinases, Brk (breast tumor kinase), and ABL2/Arg kinase . Crystal structure studies have revealed that this interaction is a canonical SH2-phosphotyrosine complex with a binding affinity (Kd) of 0.3 ± 0.1 μM . The interaction recruits ARHGAP35 to the plasma membrane and creates a signaling complex that links Ras and Rho GTPase pathways, ultimately affecting cytoskeletal dynamics, cell adhesion, and migration .

Functionally, Tyr1105 phosphorylation leads to RhoA inactivation while promoting Ras activation, providing a mechanism for coordinated regulation of these distinct GTPase pathways .

What applications are validated for Phospho-ARHGAP35 (Tyr1105) antibodies?

Commercial Phospho-ARHGAP35 (Tyr1105) antibodies have been validated for multiple applications:

These antibodies have been successfully used to detect phosphorylated ARHGAP35 in various experimental contexts, including EGF-stimulated A431 cells, where increased Tyr1105 phosphorylation can be observed .

What species reactivity can I expect from Phospho-ARHGAP35 (Tyr1105) antibodies?

The species reactivity of commercial Phospho-ARHGAP35 (Tyr1105) antibodies varies by manufacturer:

This cross-species reactivity is likely due to high conservation of the sequence surrounding Tyr1105 across mammalian species. When planning experiments with non-human samples, researchers should still confirm cross-reactivity with their specific antibody lot through appropriate validation experiments .

How should Phospho-ARHGAP35 (Tyr1105) antibodies be stored for optimal stability?

For maximum stability and retention of activity:

• Long-term storage: -20°C for up to one year from receipt

• Short-term storage: 4°C for up to one month for frequent use

• Avoid repeated freeze-thaw cycles, which can lead to antibody degradation

Most commercial antibodies are supplied in a stabilizing buffer containing:

• PBS (pH 7.2-7.4)

• 50% glycerol

• 0.02% sodium azide

• In some cases, BSA (0.5-1.0%)

To minimize freeze-thaw cycles, it is recommended to prepare small aliquots of the antibody upon receipt and thaw only what is needed for each experiment .

How can I validate the specificity of Phospho-ARHGAP35 (Tyr1105) antibodies?

To confirm that your antibody specifically recognizes ARHGAP35 phosphorylated at Tyr1105:

• Phosphatase treatment:

  • Split your protein sample into two aliquots

  • Treat one aliquot with lambda phosphatase

  • Compare signal between treated and untreated samples

  • Loss of signal in the phosphatase-treated sample confirms phospho-specificity

• Stimulation experiments:

  • Compare samples from unstimulated cells with those treated with EGF (100 ng/ml)

  • Western blots should show increased signal in EGF-treated samples

• Peptide competition:

  • Pre-incubate antibody with the phospho-peptide used as immunogen (typically containing the N-I-Y(p)-S-V sequence)

  • In parallel, pre-incubate with non-phosphorylated peptide

  • The phospho-peptide should abolish signal while non-phospho-peptide should not

• Genetic controls:

  • Use ARHGAP35 knockout/knockdown cells or tissues

  • Compare with wild-type samples

  • Absence of signal in knockout/knockdown samples confirms target specificity

• Phospho-site mutant:

  • Express wild-type ARHGAP35 and Y1105F mutant

  • Compare phospho-antibody recognition

  • Lack of signal with Y1105F mutant confirms site-specificity

What controls should I include when using Phospho-ARHGAP35 (Tyr1105) antibodies?

Essential controls for experiments with Phospho-ARHGAP35 (Tyr1105) antibodies:

For immunostaining experiments, include additional controls:

• Peptide competition (with phospho and non-phospho peptides)

• Secondary antibody only control

How can I optimize Western blot protocols for detecting phosphorylated ARHGAP35 at Tyr1105?

ARHGAP35 is a large protein (~190 kDa) and phosphorylation detection requires specific optimization:

• Sample preparation:

  • Add phosphatase inhibitors to lysis buffer

  • Keep samples cold throughout processing

  • Process samples quickly to preserve phosphorylation status

• Gel electrophoresis:

  • Use lower percentage gels (6-8%) to resolve the high molecular weight ARHGAP35

  • Consider gradient gels for better resolution

  • Load 30-50 μg total protein per lane

• Transfer:

  • Extend transfer time for complete transfer of high molecular weight proteins

  • Use wet transfer systems rather than semi-dry for large proteins

• Blocking:

  • Use 5% BSA in TBST rather than milk (milk contains phospho-proteins)

  • Block for 1 hour at room temperature or overnight at 4°C

• Antibody incubation:

  • Use recommended dilutions (typically 1:500-1:1000)

  • Incubate primary antibody overnight at 4°C

  • Wash thoroughly with TBST

Commercial antibodies from multiple vendors have successfully detected phospho-ARHGAP35 (Tyr1105) in various cell types, with A431 cells treated with EGF serving as a positive control system .

What are the key molecular interactions mediated by ARHGAP35 Tyr1105 phosphorylation?

The crystal structure of p120RasGAP N-terminal SH2 domain in complex with the phosphorylated Tyr1105 peptide reveals the molecular basis of this interaction:

• Key structural features:

  • The interaction buries 1290 Ų of total surface area

  • The peptide binds perpendicular to the central SH2 domain β-sheet

  • pTyr1105 (position 0) and Pro-1108 (position +3) insert into specific binding pockets

• Critical residue interactions:

  • pTyr1105 is coordinated by a salt-bridge to Arg-207 (conserved arginine of the FLVR motif)

  • Additional coordination occurs through Arg-188 and Ser-209

  • A three-residue cation-π stack forms between the pTyr1105 phenyl-ring, Arg-231, and Arg-212

  • Pro-1108 inserts into the specificity-determining SH2 hydrophobic pocket formed by Phe-230, Leu-262, Ile-241, and Tyr-256

• Binding affinity:

  • Isothermal titration calorimetry shows a Kd of 0.3 ± 0.1 μM

  • Mutation of the FLVR motif arginine (R207A) abolishes detectable binding

• Functional consequences:

  • This interaction recruits ARHGAP35 to the plasma membrane

  • Forms a signaling complex linking Ras and Rho GTPase pathways

  • Additional phosphorylation at Tyr1087 helps stabilize this interaction

Understanding these molecular details provides insight into how phosphorylation regulates ARHGAP35 function and may guide development of inhibitors or modulators of this interaction.

What stimulation conditions promote ARHGAP35 Tyr1105 phosphorylation in cell culture?

Several experimental conditions can induce ARHGAP35 Tyr1105 phosphorylation:

• Growth factor stimulation:

  • EGF treatment (100 ng/ml) of A431 cells is well-documented to increase Tyr1105 phosphorylation

  • Treatment duration is typically 5-30 minutes for optimal phosphorylation

• Src family kinase activation:

  • Src family kinases directly phosphorylate ARHGAP35 at Tyr1105

  • Src activators can be used to increase phosphorylation

• Cell adhesion:

  • Integrin-mediated adhesion promotes Tyr1105 phosphorylation

  • Plating cells on fibronectin or other extracellular matrix proteins can stimulate phosphorylation

• Other tyrosine kinases:

  • Brk (breast tumor kinase) and ABL2/Arg kinase have been shown to phosphorylate ARHGAP35 at Tyr1105

  • Specific activators of these kinases may be used as alternative stimulation methods

For optimal detection of phosphorylation:

• Serum-starve cells for 4-24 hours before stimulation

• Include appropriate phosphatase inhibitors in lysis buffer

• Process samples quickly to minimize phosphorylation loss

How is ARHGAP35 Tyr1105 phosphorylation regulated in different cellular contexts?

ARHGAP35 Tyr1105 phosphorylation is regulated through multiple mechanisms:

• Kinase activity:

  • Src family kinases are primary mediators of Tyr1105 phosphorylation

  • Brk (breast tumor kinase) phosphorylates Tyr1105, promoting RhoA inactivation and Ras activation

  • ABL2/Arg kinase can also phosphorylate this site, activating p190RhoGAP

• Phosphatase activity:

  • Protein tyrosine phosphatases counterbalance kinase activity

  • Temporal control of phosphorylation is achieved through balanced kinase/phosphatase action

• Cellular localization:

  • Phosphorylated ARHGAP35 typically localizes to the plasma membrane, cytoskeleton, and cilium basal body

  • Subcellular targeting influences accessibility to kinases and phosphatases

• Context-dependent regulation:

  • In gastric cancer, ARHGAP35 expression is downregulated, affecting its phosphorylation-dependent functions

  • Expression levels correlate with tumor stage and metastatic status

• Coordinated phosphorylation:

  • Tyr1087 phosphorylation works in concert with Tyr1105 to stabilize interaction with p120RasGAP

  • Multiple phosphorylation sites on ARHGAP35 create complex regulatory patterns

What is the role of ARHGAP35 Tyr1105 phosphorylation in cancer progression and metastasis?

ARHGAP35 has been identified as a tumor suppressor, with important implications for cancer biology:

• Altered expression in cancer:

  • ARHGAP35 is downregulated in gastric cancer tissues

  • Decreased expression correlates with metastatic status

• Clinical correlations in gastric cancer patients:

  Clinicopathological Variable	Total (n=83)	Weak ARHGAP35 Expression (n=48)	Strong ARHGAP35 Expression (n=35)	P value
  T stage: T1+T2	7	1	6	0.0284
  T stage: T3+T4	78	48	30
  N stage: N0+N1	32	15	17	0.0307
  N stage: N2+N3	51	36	15

  These data show significant association between ARHGAP35 expression and tumor invasion (T stage) and lymph node metastasis (N stage) .

• Functional consequences:

  • ARHGAP35 knockdown promotes cell motility in vitro

  • Phosphorylation at Tyr1105 regulates RhoA activity, which controls cytoskeletal dynamics

  • The balance between RhoA inhibition and Ras activation influences cell migration and invasion

• Therapeutic implications:

  • Monitoring Tyr1105 phosphorylation status could serve as a prognostic biomarker

  • Targeting pathways that regulate ARHGAP35 phosphorylation may have therapeutic potential

  • Restoring ARHGAP35 expression or function could suppress metastatic phenotypes

• Context-dependent effects:

  • In some cancers, ARHGAP35 acts as a tumor suppressor

  • The specific role of Tyr1105 phosphorylation may vary by cancer type and cellular context

How can I design experiments to study the spatiotemporal dynamics of ARHGAP35 Tyr1105 phosphorylation?

Advanced methodologies for spatiotemporal analysis of ARHGAP35 phosphorylation:

• Live-cell imaging approaches:

  • Use phospho-specific antibodies in fixed-time-point experiments to map phosphorylation patterns during cell migration or division

  • Apply proximity ligation assays (PLA) to visualize ARHGAP35-p120RasGAP interactions in situ

  • Combine with RhoA activity biosensors to correlate phosphorylation with functional outcomes

• Quantitative microscopy workflows:

  • Implement high-content imaging to analyze phosphorylation patterns across cell populations

  • Measure phosphorylation gradients during directed cell migration

  • Quantify co-localization with adhesion components and RhoA activity zones

• Temporal regulation analysis:

  • Design pulse-chase experiments with stimulators/inhibitors of phosphorylation

  • Use time-course studies following EGF stimulation (100 ng/ml) to capture phosphorylation dynamics

  • Correlate phosphorylation timing with cellular events (adhesion formation/disassembly)

• Tissue-level analysis:

  • Apply phospho-specific IHC to study ARHGAP35 phosphorylation in tissue sections

  • Compare phosphorylation patterns at tumor invasion fronts versus tumor cores

  • Correlate with clinical outcomes and metastatic status as observed in gastric cancer studies

• Molecular interaction studies:

  • Use the crystal structure information of the p120RasGAP-p190RhoGAP interaction to design experiments that probe specific residues

  • Create phospho-mimetic or phospho-dead mutants to study functional consequences

These approaches enable researchers to understand when and where ARHGAP35 phosphorylation occurs during cellular processes, providing insights into its regulatory mechanisms.

What are the challenges in detecting endogenous phosphorylated ARHGAP35 compared to overexpression systems?

Detection of endogenous phosphorylated ARHGAP35 presents several challenges:

• Abundance issues:

  • Endogenous phosphorylated ARHGAP35 may be present at low levels

  • Signal-to-noise ratio can be problematic, especially in unstimulated conditions

  • May require signal enhancement techniques or enrichment by immunoprecipitation

• Phosphorylation lability:

  • Phosphorylated tyrosine residues are rapidly dephosphorylated during sample processing

  • Requires stringent phosphatase inhibitor use and rapid processing

  • Sample handling conditions are critical for preserving phosphorylation status

• Antibody sensitivity and specificity:

  • Commercial antibodies vary in their ability to detect endogenous levels

  • Most validation is performed in overexpression systems or strongly stimulated cells (e.g., EGF-treated A431 cells)

  • Background binding can interfere with detection of low-abundance endogenous protein

• Experimental approaches to overcome these challenges:

  • Use stimulation conditions that maximize phosphorylation (EGF treatment, 100 ng/ml)

  • Enrich phosphorylated proteins through immunoprecipitation before detection

  • Implement tyramide signal amplification for immunofluorescence applications

  • Include comprehensive controls to verify specificity (phosphatase treatment, knockdown/knockout samples)

• Cell type considerations:

  • ARHGAP35 expression varies across cell types

  • Gastric cancer cells show variable ARHGAP35 expression levels correlated with clinical parameters

  • Choose appropriate cellular models with sufficient endogenous expression

How do Tyr1087 and Tyr1105 phosphorylation sites interact to regulate ARHGAP35 function?

ARHGAP35 contains multiple phosphorylation sites that work in concert:

• Coordinated regulation:

  • Both Tyr1105 and Tyr1087 are involved in the association of ARHGAP35 with p120RasGAP

  • Phosphorylation of Tyr1105 is essential for complex formation

  • Additional phosphorylation of Tyr1087 helps to stabilize the interaction

• Structural basis:

  • Crystal structure studies have focused on the Tyr1105 interaction with the N-terminal SH2 domain of p120RasGAP

  • The relative spatial arrangement of these phosphorylation sites likely facilitates optimal binding

• Kinase specificity:

  • Both sites can be phosphorylated by Src family kinases

  • Commercial antibodies are available for detecting each phosphorylation site specifically

  • Different kinases may preferentially target one site over the other

• Functional consequences:

  • The dual phosphorylation may provide more robust regulation of ARHGAP35 localization and activity

  • This creates potential for fine-tuning of signaling through differential phosphorylation

  • The combined effect likely enhances the stability of the ARHGAP35-p120RasGAP complex

• Experimental approaches:

  • Use site-specific phospho-antibodies to monitor each site independently

  • Create single and double phosphorylation site mutants to assess their individual and combined roles

  • Compare the binding affinities of singly and doubly phosphorylated peptides to p120RasGAP

Understanding the interplay between these phosphorylation sites provides insight into the complex regulation of ARHGAP35 function in normal and pathological states.

How can multi-parameter analyses with Phospho-ARHGAP35 (Tyr1105) antibodies advance our understanding of signaling networks?

Integrating Phospho-ARHGAP35 (Tyr1105) detection with other signaling pathway markers:

• Co-detection strategies:

  • Combine phospho-ARHGAP35 staining with markers for:

    • Active RhoA (RhoA-GTP)

    • p120RasGAP localization

    • Phosphorylated Src family kinases

    • Focal adhesion components

    • Actin cytoskeletal structures

  • This reveals functional relationships between phosphorylation and downstream effectors

• Clinical correlations:

  • In gastric cancer tissues, ARHGAP35 expression correlates with T stage and N stage

  • Multi-parameter analysis can reveal whether phosphorylation status provides additional prognostic information

  • Can help identify patient subgroups that might benefit from targeted therapies

• Pathway integration:

  • ARHGAP35 phosphorylation links Ras and Rho signaling pathways

  • Multi-parameter analysis can reveal how these pathways are coordinated

  • Provides insight into crosstalk mechanisms between different GTPase-regulated pathways

• Therapeutic implications:

  • Identify key nodes in signaling networks that could be targeted therapeutically

  • Understand resistance mechanisms in existing therapies

  • Develop combination strategies based on network understanding

• Technical approaches:

  • Multiplexed immunofluorescence with spectral unmixing

  • Sequential immunohistochemistry on tissue sections

  • Combined proteomic and phospho-proteomic analyses

  • Correlation with functional assays (migration, invasion, proliferation)

By implementing these multi-parameter approaches, researchers can gain a systems-level understanding of how ARHGAP35 Tyr1105 phosphorylation integrates with broader signaling networks to regulate cellular behavior in normal and pathological contexts.