Phospho-ARHGAP35 (Y1087) Antibody

What is ARHGAP35 and what cellular functions does it regulate?

ARHGAP35, also known as GRF-1, GRLF1, or p190RhoGAP, is a Rho GTPase-activating protein with a molecular weight of approximately 170 kDa. It functions as a key regulator of cellular processes including cytoskeletal organization, cell migration, adhesion, and polarity establishment . The protein plays a crucial role in signaling pathways as a negative regulator of RhoA activity, thereby modulating actin dynamics during various cellular processes. Its phosphorylation at specific residues, including tyrosine 1087, serves as a molecular switch controlling its activity and interactions with other signaling molecules . In research contexts, ARHGAP35 has been implicated in cancer progression pathways, making it a target of interest for understanding metastatic behavior and potential therapeutic interventions.

What are the key characteristics of Phospho-ARHGAP35 (Y1087) Antibody?

Phospho-ARHGAP35 (Y1087) Antibody is a rabbit polyclonal antibody that specifically recognizes the phosphorylated form of ARHGAP35 at tyrosine residue 1087 . The antibody is typically produced by immunizing rabbits with a synthesized phospho-peptide corresponding to the region surrounding the phosphorylation site Y1087 of human GRF-1 . It exhibits cross-reactivity across human, mouse, and rat species, making it versatile for comparative studies . The antibody is formulated as a liquid in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide . It has been validated for Western blotting (recommended dilution 1:500-1:2000) and ELISA (recommended dilution 1:10000) applications . The specificity of this antibody allows researchers to detect endogenous levels of GRF-1 protein only when phosphorylated at Y1087, providing a powerful tool for investigating phosphorylation-dependent signaling events.

How does phosphorylation at Y1087 affect ARHGAP35 function?

Phosphorylation at Y1087 of ARHGAP35 represents one of several key regulatory phosphorylation events that modulate this protein's function in signal transduction pathways. While the specific effects of Y1087 phosphorylation are still being elucidated, the broader context of ARHGAP35 phosphorylation provides insight into potential mechanisms. Related phosphorylation events on ARHGAP35 have documented effects: phosphorylation at Tyr-1105 by PTK6 promotes association with RASA1, resulting in RHOA inactivation while activating RAS signaling . Phosphorylation at Tyr-308 by PDGFRA inhibits binding to GTF2I . Phosphorylation by MAPK1 and MAPK3 at the C-terminal region inhibits GAP function and affects protein localization during focal adhesion formation . These findings suggest that Y1087 phosphorylation likely plays a specific role in regulating ARHGAP35's interactions with binding partners and/or its enzymatic activity, potentially affecting downstream cytoskeletal organization and cellular behaviors.

What are the optimal experimental conditions for detecting Phospho-ARHGAP35 (Y1087) in different cell types?

Detecting Phospho-ARHGAP35 (Y1087) requires careful optimization of experimental conditions based on cell type and experimental question. For Western blot analysis, the following methodology has been validated:

• Sample preparation: Lyse cells in a buffer containing phosphatase inhibitors to preserve phosphorylation status. For adherent cells like 293T cells, direct lysis on plate after appropriate stimulation yields good results .

• Sample loading: Load 20-40 μg of total protein per lane on 8% SDS-PAGE gels to provide sufficient resolution for this high molecular weight protein (~170 kDa).

• Transfer conditions: Use wet transfer (100V for 90 minutes) to PVDF membranes for optimal detection of high molecular weight proteins.

• Blocking: 5% BSA in TBST is preferred over milk-based blocking buffers which contain phosphatases that might reduce signal.

• Antibody incubation: Primary antibody dilution of 1:1000 has been validated for 293T and K562 cells , but optimization between 1:500-1:2000 may be necessary for other cell types.

• Detection system: Enhanced chemiluminescence with exposure times ranging from 30 seconds to 5 minutes depending on expression levels.

For immunoprecipitation experiments, higher antibody concentrations (1:100-1:500) may be required, along with extended incubation periods (overnight at 4°C) to efficiently pull down the phosphorylated protein.

How can Phospho-ARHGAP35 (Y1087) Antibody be used to study cross-talk between different signaling pathways?

Phospho-ARHGAP35 (Y1087) Antibody provides a valuable tool for investigating signaling pathway cross-talk due to ARHGAP35's position at the intersection of multiple regulatory cascades. Methodological approaches for such studies include:

• Sequential immunoprecipitation: First immunoprecipitate with phospho-ARHGAP35 (Y1087) antibody, then probe for interacting partners or other post-translational modifications to identify multi-pathway regulation.

• Comparative phosphorylation analysis: Stimulate cells with different pathway activators (e.g., growth factors, cytokines) and compare Y1087 phosphorylation patterns alongside other ARHGAP35 phosphorylation sites (e.g., Y1105, Y308, S1221, T1226) to map pathway-specific phosphorylation signatures.

• Inhibitor studies: Use specific pathway inhibitors (e.g., for Src family kinases, MAPK, GSK3β) to determine which upstream pathways regulate Y1087 phosphorylation. This approach is particularly informative since ARHGAP35 is known to be phosphorylated by multiple kinases including FYN, SRC, MAPK1, MAPK3, and GSK3B at different sites .

• Co-localization experiments: Combine Phospho-ARHGAP35 (Y1087) antibody with markers for specific cellular compartments or signaling complexes to determine how Y1087 phosphorylation affects protein localization and complex formation.

These approaches can reveal how ARHGAP35 phosphorylation at Y1087 might serve as a convergence point for different signaling inputs or diversification point for multiple downstream pathways.

What are the latest findings regarding the role of Y1087 phosphorylation in cancer biology?

Recent research utilizing Phospho-ARHGAP35 (Y1087) antibodies has contributed to understanding this specific phosphorylation event in cancer progression. While the complete picture continues to emerge, several findings highlight its significance:

• Altered Y1087 phosphorylation patterns have been observed in various cancer cell lines, particularly those with invasive phenotypes, suggesting a potential role in metastatic behavior.

• Correlation studies have identified associations between Y1087 phosphorylation status and Rho GTPase signaling perturbations in tumor samples, consistent with ARHGAP35's known role as a regulator of cell migration and invasion through its GAP activity.

• The phosphorylation at Y1087 appears to have context-dependent effects on ARHGAP35's function as either a tumor suppressor or promoter, potentially by altering its interaction with different binding partners in specific cellular contexts.

These findings build on our understanding of ARHGAP35 as a multifunctional protein involved in cancer-related processes. While Y1087 phosphorylation specifically represents one regulatory mechanism, it should be considered alongside other post-translational modifications that collectively determine ARHGAP35's contribution to cancer cell phenotypes.

What controls should be included when using Phospho-ARHGAP35 (Y1087) Antibody in Western blot experiments?

A robust experimental design for Western blot analysis using Phospho-ARHGAP35 (Y1087) Antibody should include the following controls:

• Positive control: Lysates from cells known to express phosphorylated ARHGAP35, such as K562 cells, which have been validated in previous studies .

• Negative control: Include one of the following:

  • Lysates from the same cell type treated with phosphatase

  • Lysates from cells where ARHGAP35 has been knocked down via siRNA or CRISPR

  • Lysates from cells treated with kinase inhibitors that prevent Y1087 phosphorylation

• Loading control: A housekeeping protein such as GAPDH or β-actin to confirm equal protein loading across samples.

• Phosphorylation specificity control: When available, include a blocking peptide competition assay. Pre-incubate the antibody with the phosphorylated immunogen peptide to demonstrate signal specificity.

• Total protein control: Probe parallel blots or strip and reprobe with antibodies against total ARHGAP35 to determine changes in phosphorylation versus total protein levels.

• Molecular weight markers: Include to confirm the detection of a band at the expected molecular weight of approximately 170 kDa.

Including these controls allows for proper interpretation of results and provides confidence in the specificity of the observed signals.

How can phosphorylation at Y1087 be induced or inhibited for experimental purposes?

Manipulating phosphorylation at Y1087 of ARHGAP35 can be achieved through several experimental approaches:

Induction methods:

• Growth factor stimulation: Treat cells with growth factors that activate tyrosine kinases. While specific kinases for Y1087 are not fully characterized, platelet-derived growth factor (PDGF) treatment has been shown to induce phosphorylation at other tyrosine residues of ARHGAP35 .

• Cell adhesion: Allow cells to spread on fibronectin or other extracellular matrix components, which activates integrin signaling and subsequent tyrosine phosphorylation events.

• Phosphatase inhibitor treatment: Apply sodium orthovanadate (1-2 mM) or phenylarsine oxide (5-10 μM) to cells to inhibit tyrosine phosphatases, enhancing detection of phosphorylated proteins.

Inhibition methods:

• Kinase inhibitors: Src family kinase inhibitors (PP2, dasatinib) may reduce Y1087 phosphorylation, as FYN and SRC are known to phosphorylate ARHGAP35 in brain tissues .

• Serum starvation: Culture cells in serum-free medium for 16-24 hours to reduce baseline phosphorylation.

• Genetic approaches: Express a Y1087F mutant version of ARHGAP35 that cannot be phosphorylated at this site.

• Pharmacological disruption: Cytoskeletal disruptors like cytochalasin D may indirectly affect Y1087 phosphorylation by altering cell adhesion and associated signaling events.

These approaches provide experimental tools to interrogate the functional significance of Y1087 phosphorylation in different cellular contexts.

What is the recommended protocol for Phospho-ELISA using Phospho-ARHGAP35 (Y1087) Antibody?

For conducting a Phospho-ELISA to detect and quantify ARHGAP35 Y1087 phosphorylation, the following protocol is recommended based on validated approaches:

Materials required:

• Phospho-ARHGAP35 (Y1087) Antibody

• Coating buffer (usually carbonate-bicarbonate buffer, pH 9.6)

• Blocking solution (5% BSA in PBS)

• Wash buffer (0.05% Tween-20 in PBS)

• Sample dilution buffer (1% BSA in PBS)

• HRP-conjugated secondary antibody

• TMB substrate solution

• Stop solution (2N H₂SO₄)

• 96-well ELISA plate

Protocol:

• Plate preparation:

  • Coat ELISA plate wells with capture antibody against total ARHGAP35 diluted in coating buffer (1:1000)

  • Incubate overnight at 4°C

  • Wash 3 times with wash buffer

• Blocking:

  • Add 300 μl blocking solution to each well

  • Incubate for 2 hours at room temperature

  • Wash 3 times with wash buffer

• Sample addition:

  • Add 100 μl of cell lysate samples to wells

  • Incubate for 2 hours at room temperature

  • Wash 5 times with wash buffer

• Detection antibody:

  • Add 100 μl of Phospho-ARHGAP35 (Y1087) Antibody diluted 1:10000 in sample buffer

  • Incubate overnight at 4°C

  • Wash 5 times with wash buffer

• Secondary antibody:

  • Add 100 μl of HRP-conjugated anti-rabbit IgG (1:5000)

  • Incubate for 1 hour at room temperature

  • Wash 7 times with wash buffer

• Signal development:

  • Add 100 μl TMB substrate solution

  • Incubate in dark for 15-30 minutes

  • Add 50 μl stop solution

• Measurement:

  • Read absorbance at 450 nm with reference at 620 nm

This protocol has been validated for comparing phosphorylation levels between different experimental conditions, as shown in validation data where phospho-peptide immunogen and non-phosphopeptide controls were clearly distinguished .

How can researchers distinguish between true phospho-specific signals and non-specific binding?

Differentiating true phospho-specific signals from non-specific binding requires systematic analytical approaches:

• Molecular weight verification:
  Examine whether the detected band appears at the expected molecular weight of ARHGAP35 (~170 kDa). Multiple bands or bands at unexpected molecular weights may indicate non-specific binding.

• Phosphatase treatment control:
  Divide your sample into two portions and treat one with lambda phosphatase. The phospho-specific signal should disappear or significantly decrease in the treated sample while non-specific signals will remain unchanged.

• Peptide competition assay:
  Pre-incubate the antibody with the phosphorylated peptide immunogen and separately with the corresponding non-phosphorylated peptide. A true phospho-specific signal will be blocked by the phosphorylated peptide but not by the non-phosphorylated version, as demonstrated in phospho-ELISA validation data .

• Phosphorylation induction:
  Compare samples from untreated cells versus cells treated with agents known to induce ARHGAP35 phosphorylation. A phospho-specific signal should increase with treatments that enhance phosphorylation.

• Concentration gradient analysis:
  Test different antibody dilutions (1:500, 1:1000, 1:2000) to identify the optimal concentration that maximizes specific signal while minimizing background .

• Multiple detection methods:
  Confirm phosphorylation using complementary approaches (Western blot, immunoprecipitation, and ELISA) as each method has different specificity profiles .

By applying these analytical approaches, researchers can confidently distinguish phospho-specific signals from non-specific background.

What factors might affect the detection sensitivity of Phospho-ARHGAP35 (Y1087) in different experimental systems?

Several experimental factors can significantly impact detection sensitivity when working with Phospho-ARHGAP35 (Y1087) Antibody:

• Sample preparation factors:

  • Phosphatase inhibitor cocktail composition and concentration

  • Cell lysis buffer formulation (RIPA vs. NP-40 vs. Triton X-100)

  • Sample handling time and temperature

  • Freeze-thaw cycles (should be minimized to preserve phosphorylation)

• Protein expression levels:

  • Basal ARHGAP35 expression varies across cell types

  • Phosphorylation stoichiometry may be low under certain conditions

  • Competition from other phosphorylated residues nearby

• Technical parameters:

  • Membrane type (PVDF typically outperforms nitrocellulose for phospho-proteins)

  • Blocking agent (BSA preferred over milk, which contains phosphatases)

  • Incubation temperature and duration

  • Antibody dilution optimization (1:500-1:2000 range recommended)

  • Detection system sensitivity (enhanced chemiluminescence vs. fluorescence)

• Biological variables:

  • Cell confluence and passage number

  • Growth conditions and serum levels

  • Cell cycle phase (which may affect phosphorylation status)

  • Cell stress responses that alter phosphorylation patterns

• Storage considerations:

  • Antibody stability (avoid repeated freeze-thaw cycles)

  • Storage temperature (-20°C for long-term, 4°C for up to one month)

  • Buffer composition (50% glycerol helps maintain antibody activity)

Understanding and controlling these variables is essential for achieving consistent and sensitive detection of Y1087 phosphorylation across different experimental systems.

How should conflicting results between Western blot and ELISA for Phospho-ARHGAP35 (Y1087) be reconciled?

When faced with discrepancies between Western blot and ELISA results for Phospho-ARHGAP35 (Y1087), researchers should undertake a systematic investigation:

• Technique-specific considerations:

  Parameter	Western Blot	ELISA	Reconciliation Strategy
  Sensitivity	Moderate (depends on exposure)	High	ELISA may detect lower levels of phosphorylation invisible by Western blot
  Specificity	High (size-based separation)	Variable (depends on capture antibody)	Confirm size by Western blot even when ELISA is positive
  Denaturation	Complete (SDS)	Minimal (native proteins)	Epitope accessibility may differ between methods
  Quantification	Semi-quantitative	Quantitative	Use Western blot for confirmation, ELISA for quantification

• Methodological approach to reconciliation:

  • Verify antibody batch consistency between techniques

  • Optimize antibody concentration independently for each technique (1:500-1:2000 for WB, 1:10000 for ELISA)

  • Test phosphorylated and non-phosphorylated control samples in both assays

  • Evaluate epitope accessibility in both formats

  • Consider using immunoprecipitation followed by Western blot as a third method

• Data interpretation guidelines:

  • If Western blot is positive but ELISA negative: Check ELISA coating antibody efficacy and epitope masking

  • If ELISA is positive but Western blot negative: Verify phospho-specificity using peptide competition in ELISA and consider sensitivity limitations in Western blot

  • If signal strength differs proportionally: Likely reflects inherent sensitivity differences between techniques

• Validation strategy:

  • Implement biological controls (kinase activators/inhibitors) to create samples with defined phosphorylation states

  • Use genetic approaches (Y1087F mutants) as definitive negative controls

  • Consider mass spectrometry as an orthogonal technique for phosphorylation site verification

What are emerging applications for Phospho-ARHGAP35 (Y1087) Antibody in cancer research?

Emerging applications of Phospho-ARHGAP35 (Y1087) Antibody in cancer research span multiple areas of investigation:

• Biomarker development:
  The phosphorylation status of ARHGAP35 at Y1087 may serve as a predictive or prognostic biomarker in specific cancer types, particularly those characterized by aberrant Rho GTPase signaling and metastatic potential. The antibody enables researchers to evaluate this phosphorylation event in patient-derived samples through techniques like tissue microarray analysis and immunohistochemistry.

• Therapeutic target validation:
  As understanding of ARHGAP35 regulation grows, the Y1087 phosphorylation site may emerge as a potential therapeutic target. The antibody provides a critical tool for validating target engagement and pathway modulation in drug discovery programs targeting kinases that phosphorylate this residue or phosphatases that regulate its dephosphorylation.

• Resistance mechanism studies:
  Phosphorylation at Y1087 may contribute to resistance mechanisms against targeted therapies that affect cytoskeletal dynamics or cell migration. The antibody enables monitoring of adaptive phosphorylation changes that occur in response to treatment.

• Precision medicine approaches:
  Understanding how Y1087 phosphorylation status correlates with treatment responses could inform patient stratification strategies for therapies targeting pathways that intersect with ARHGAP35 function.

These emerging applications highlight the value of Phospho-ARHGAP35 (Y1087) Antibody as a tool for advancing cancer research beyond basic mechanistic studies toward clinically relevant applications.

How might single-cell analysis techniques utilize Phospho-ARHGAP35 (Y1087) Antibody?

Single-cell analysis techniques offer powerful approaches for understanding heterogeneity in ARHGAP35 phosphorylation within complex tissues and cell populations:

• Single-cell phospho-flow cytometry:

  • Protocol adaptation: Optimize fixation (paraformaldehyde 2-4%) and permeabilization (methanol or saponin-based) conditions for intracellular staining with Phospho-ARHGAP35 (Y1087) Antibody

  • Application: Map phosphorylation patterns across different cell populations in heterogeneous samples such as tumor biopsies or mixed cell cultures

  • Advanced approach: Combine with markers for cell cycle, differentiation state, or other signaling pathways to create multidimensional phosphorylation profiles

• Mass cytometry (CyTOF):

  • Metal conjugation: Label Phospho-ARHGAP35 (Y1087) Antibody with rare earth metals

  • Multiplexing capacity: Simultaneously analyze Y1087 phosphorylation alongside dozens of other proteins and phosphorylation sites

  • Data analysis: Apply dimensionality reduction and clustering algorithms to identify cell populations with distinct phosphorylation signatures

• Imaging mass cytometry:

  • Spatial context: Preserve tissue architecture while measuring Y1087 phosphorylation at subcellular resolution

  • Colocalization analysis: Examine relationships between Y1087 phosphorylation and subcellular structures or signaling hubs

• Single-cell Western blotting:

  • Microfluidic platforms: Separate proteins from individual cells and probe for Phospho-ARHGAP35 (Y1087)

  • Quantification: Measure precise phosphorylation levels in rare cell populations

These emerging techniques extend the utility of Phospho-ARHGAP35 (Y1087) Antibody beyond conventional bulk analyses to reveal cell-to-cell variation in phosphorylation status and its relationship to cellular phenotypes and behaviors.