Phospho-ARHGDIA (S174) Antibody

What is ARHGDIA and what role does its phosphorylation at S174 play in cellular signaling?

ARHGDIA (Rho GDP-dissociation inhibitor alpha, also known as RhoGDIα) is a regulatory protein that plays a critical role in controlling Rho GTPase signaling. It functions by inhibiting the dissociation of GDP from Rho family members, thereby maintaining these factors in an inactive state . The protein actively participates in recycling and distributing activated Rho GTPases within the cell and mediates extraction from membranes of both inactive and activated molecules due to its exceptionally high affinity for prenylated forms .

Phosphorylation at Serine 174 (S174) represents a specific post-translational modification that likely influences ARHGDIA's interaction with Rho GTPases. The Phospho-ARHGDIA (S174) Antibody specifically detects ARHGDIA only when phosphorylated at this serine residue, making it an essential tool for studying this regulatory mechanism .

What applications are validated for Phospho-ARHGDIA (S174) Antibody?

The Phospho-ARHGDIA (S174) Antibody has been validated for multiple research applications:

The antibody has been validated using various experimental systems, including HUVEC cells treated with EGF and human cervix carcinoma tissues .

What is the specificity profile of the Phospho-ARHGDIA (S174) Antibody?

The Phospho-ARHGDIA (S174) Antibody specifically detects endogenous levels of ARHGDIA protein only when phosphorylated at Serine 174 . This specificity has been demonstrated through:

• Phospho-ELISA assays comparing binding to phosphorylated versus non-phosphorylated peptides

• Blocking experiments using the phospho-peptide immunogen

• Western blot analysis showing differential detection in stimulated versus non-stimulated cells

The antibody cross-reacts with phosphorylated ARHGDIA from human, mouse, and rat samples, indicating conservation of this phosphorylation site across these species .

What is the optimal storage and handling protocol for Phospho-ARHGDIA (S174) Antibody?

For optimal antibody performance, follow these storage and handling guidelines:

• Long-term storage: Store at -20°C for up to one year

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

• Avoid repeated freeze-thaw cycles by preparing small aliquots upon receipt

• The antibody is typically provided in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide

• Always centrifuge briefly before opening the vial to ensure solution homogeneity

What controls should be included when using the Phospho-ARHGDIA (S174) Antibody?

To ensure experimental validity, incorporate these controls:

• Positive control: HUVEC cells treated with EGF (200ng/ml for 30 minutes) show detectable phosphorylation at S174

• Negative control: Pre-incubation of the antibody with the phosphorylated immunogen peptide should abolish the signal

• Loading control: When performing western blots, include detection of total ARHGDIA or housekeeping proteins like GAPDH

• Specificity control: Compare detection in samples treated with and without phosphatase

How does phosphorylation at S174 compare with other ARHGDIA phosphorylation sites?

ARHGDIA contains multiple phosphorylation sites that may have distinct or overlapping functions. Research findings provide a basis for comparison:

S101 Phosphorylation:

• Increases by 40% in response to insulin stimulation in skeletal muscle cells

• Associated with dissociation of the Rac1-RhoGDIα complex

• Non-phosphorylatable S101A mutant represses both basal and insulin-stimulated Rac1 activity

• Affects GLUT4 translocation in muscle cells

S174 Phosphorylation:

• Detected in HUVEC cells following EGF treatment

• Located in the amino acid region 140-189 of human ARHGDIA

To systematically compare these sites, researchers should:

• Examine phosphorylation dynamics under various stimuli

• Create and test phospho-mimetic (S→D) and non-phosphorylatable (S→A) mutants

• Assess differential effects on Rho GTPase binding affinity and activity

• Investigate potential synergistic or antagonistic relationships between multiple phosphorylation events

What are the experimental approaches to determine kinases responsible for S174 phosphorylation?

Identifying the kinase(s) responsible for S174 phosphorylation requires systematic investigation:

• Kinase Prediction Analysis:

  • Analyze the sequence surrounding S174 for consensus phosphorylation motifs

  • Consider that insulin signaling affects RhoGDIα phosphorylation at S101

  • Evaluate PKA as a potential candidate, given its role in phosphorylating other cytoskeletal regulators

• Pharmacological Approaches:

  • Treat cells with specific kinase activators (e.g., forskolin for PKA pathway)

  • Apply selective kinase inhibitors to identify pathways involved

  • Use EGF treatment, which has been shown to induce S174 phosphorylation

• Genetic Manipulation:

  • Perform siRNA knockdown of candidate kinases

  • Express constitutively active or dominant-negative kinase mutants

  • Generate phosphorylation-site mutants (S174A) to confirm specificity

• Biochemical Validation:

  • Conduct in vitro kinase assays with purified components

  • Perform immunoprecipitation followed by mass spectrometry to identify associated kinases

  • Use phospho-specific antibodies to monitor phosphorylation dynamics

How can researchers validate the functional consequences of ARHGDIA S174 phosphorylation?

To establish the functional significance of ARHGDIA S174 phosphorylation:

• Protein Interaction Studies:

  • Compare binding affinities of wild-type, phospho-mimetic (S174D/E), and non-phosphorylatable (S174A) ARHGDIA to different Rho GTPases

  • Perform co-immunoprecipitation studies under conditions that promote or inhibit S174 phosphorylation

  • Use FRET-based approaches to monitor interaction dynamics in live cells

• Subcellular Localization Analysis:

  • Examine the distribution of phosphorylated ARHGDIA versus total ARHGDIA

  • Monitor redistribution following stimuli that induce S174 phosphorylation

  • Compare localization patterns of phospho-mimetic and non-phosphorylatable mutants

• Functional Readouts:

  • Assess effects on Rho GTPase activation using pull-down assays

  • Examine cytoskeletal reorganization through F-actin staining

  • Measure cell migration, invasion, or adhesion dynamics

  • Evaluate endothelial barrier function, which may be regulated by Rho GTPases

  • Investigate GLUT4 translocation, which is affected by RhoGDIα phosphorylation at S101

• Systems-Level Analysis:

  • Perform transcriptomic or proteomic profiling in cells expressing wild-type versus mutant ARHGDIA

  • Identify pathways and processes affected by altered S174 phosphorylation status

What are the methodological considerations for detecting phospho-ARHGDIA (S174) in different sample types?

Optimal detection strategies vary by sample type and experimental context:

Cell Lysates (Western Blotting):

• Include phosphatase inhibitors during sample preparation

• Use fresh samples or store at -80°C with protease inhibitors

• Recommended dilution: 1:500-1:2000

• Positive control: HUVEC cells treated with EGF (200ng/ml, 30 minutes)

Tissue Sections (Immunohistochemistry):

• Paraffin-embedded or frozen sections both compatible

• Antigen retrieval methods may enhance detection

• Recommended dilution: 1:100-1:300

• Validated in human cervix carcinoma tissue

ELISA Applications:

• Recommended dilution: 1:20000

• Can be used for quantitative analysis of phosphorylation levels

• Include a standard curve with recombinant phosphorylated protein

How can phospho-ARHGDIA (S174) analysis be integrated into broader Rho GTPase signaling studies?

A comprehensive investigation of Rho GTPase signaling should integrate multiple analytical approaches:

• Coordinated Analysis of GTPase Activation and GDI Phosphorylation:

  • Perform active GTPase pull-down assays (using GST-RBD/PBD domains)

  • Simultaneously assess ARHGDIA phosphorylation status

  • Correlate temporal dynamics of both events following stimulation

• Multi-parameter Microscopy:

  • Co-stain for phospho-ARHGDIA (S174), total ARHGDIA, active GTPases, and downstream effectors

  • Analyze subcellular co-localization patterns

  • Implement super-resolution techniques for detailed spatial information

• Functional Correlation Studies:

  • Measure cytoskeletal reorganization (stress fiber formation)

  • Assess changes in cellular processes regulated by Rho GTPases:

    • Cell migration and invasion

    • Endothelial barrier function

    • Glucose transport via GLUT4 translocation

• Manipulation of the Pathway:

  • Express phospho-mimetic or non-phosphorylatable ARHGDIA mutants

  • Use constitutively active or dominant-negative Rho GTPase mutants

  • Apply specific inhibitors of Rho GTPases (e.g., C3 transferase for RhoA)

  • Modulate upstream kinases that phosphorylate ARHGDIA

What are the potential pathophysiological implications of altered ARHGDIA S174 phosphorylation?

Understanding ARHGDIA phosphorylation in disease contexts provides research opportunities:

Metabolic Disorders:

• RhoGDIα phosphorylation at S101 affects insulin-stimulated Rac1 activation and GLUT4 translocation in skeletal muscle

• S174 phosphorylation might similarly impact glucose metabolism or insulin signaling

Kidney Disease:

• Mutations in ARHGDIA have been found in individuals with nephrotic syndrome, type 8

• Phosphorylation status may affect disease progression or response to therapy

Cancer:

• ARHGDIA expression may be altered in tumors

• Phosphorylation could modify its interaction with Rho GTPases, affecting cell migration and invasion

• Immunohistochemistry has validated phospho-ARHGDIA (S174) detection in cervix carcinoma

Vascular Disorders:

• Rho GTPases regulate endothelial barrier function

• ARHGDIA phosphorylation might influence vascular permeability in inflammatory conditions

Research approaches should include:

• Analyzing phosphorylation levels in patient samples versus controls

• Correlating phosphorylation status with disease progression

• Developing animal models with phosphorylation-site mutations

• Screening for compounds that modulate ARHGDIA phosphorylation