Phospho-ITGB3 (Tyr773) Antibody

What is ITGB3 and why is its phosphorylation at Tyr773 significant?

ITGB3 (Integrin beta 3) is a glycoprotein also known as GPIIIa or CD61 that functions as the beta subunit of several integrin complexes, including the platelet membrane adhesive protein receptor complex GP IIb/IIIa. The protein weighs approximately 87 kDa and serves as a critical component in cell adhesion and signaling pathways .

Phosphorylation of ITGB3 at tyrosine 773 represents a key post-translational modification that regulates its biological activity. This specific phosphorylation:

• Forms part of a regulatory mechanism in integrin-mediated signaling

• Plays a critical role in endothelial cell migration when ITGB3 interacts with PTPRZ1 and PTN

• Contributes to cell proliferation and invasion processes in various cell types

• Serves as a potential biomarker in cancer metastasis research

The phosphorylation state at Tyr773 can significantly alter ITGB3's interactions with other cellular proteins and influence downstream signaling cascades, making it an important target for antibody-based detection in research settings.

How do I select the appropriate Phospho-ITGB3 (Tyr773) antibody for my experiments?

Selection of an appropriate Phospho-ITGB3 (Tyr773) antibody requires consideration of several factors:

Species reactivity:

• Verify the antibody's reactivity with your experimental species. Most commercial antibodies react with human, mouse, and rat ITGB3 .

• For cross-species applications, check sequence homology between your species of interest and the immunogen sequence .

Application compatibility:

Validation evidence:

• Review validation images showing specificity, such as elimination of signal in site-directed mutants (Y773F)

• Check for antibody validation using phosphopeptide competition assays

• Confirm specificity for the phosphorylated form versus the non-phosphorylated protein

For optimal results, select antibodies specifically validated for your application of interest with demonstrated specificity for the phosphorylated Tyr773 epitope.

What are the optimal protocols for detecting Phospho-ITGB3 (Tyr773) in Western blot applications?

Sample Preparation:

• Use RIPA lysis buffer for homogenization of cell or tissue samples

• Include phosphatase inhibitors (e.g., sodium orthovanadate, sodium fluoride) to preserve phosphorylation states

• Process samples quickly and maintain at 4°C throughout

Electrophoresis Conditions:

• Use 5-20% gradient SDS-PAGE gels for optimal resolution of ITGB3 (~87-100 kDa)

• Load 30 μg of protein per lane under reducing conditions

• Run at 70V (stacking gel) followed by 90V (resolving gel) for 2-3 hours

Protein Transfer:

• Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes

• Verify transfer efficiency with Ponceau S staining

Antibody Incubation:

• Block membrane with 5% non-fat milk in TBS for 1.5 hours at room temperature

• Incubate with primary antibody (0.5-2 μg/mL) overnight at 4°C

• Wash thoroughly with TBS-0.1% Tween (3 × 5 minutes)

• Incubate with appropriate HRP-conjugated secondary antibody (1:5000) for 1.5 hours at room temperature

Detection:

• Develop using enhanced chemiluminescence (ECL) detection system

• Expected band size: 87-100 kDa (may appear larger due to glycosylation)

Critical Controls:

• Include phosphopeptide competition controls to confirm specificity

• Compare wild-type cells with cells expressing Y773F mutant ITGB3

• Include positive controls (e.g., activated platelets or endothelial cells)

How does ITGB3 Tyr773 phosphorylation differ from phosphorylation at other sites such as Tyr785?

Phosphorylation of ITGB3 occurs at multiple tyrosine residues, with Tyr773 and Tyr785 being among the most studied sites. These distinct phosphorylation events trigger different downstream effects:

Tyr773 Phosphorylation:

• Associated with endothelial cell migration through interaction with PTPRZ1 and PTN

• Involves a complex with specific signaling partners

• Antibodies against this site specifically recognize the phosphopeptide sequence P-L-Y(p)-K-E

• Detected in human, mouse, and rat samples using specific antibodies

Tyr785 Phosphorylation:

• Different signaling outcomes compared to Tyr773 phosphorylation

• Can be specifically detected using phospho-specific antibodies like A00587Y785

• Often associated with different cellular responses

• Detection can be validated through phosphopeptide competition assays

Comparative Analysis:

Research suggests these phosphorylation events may be independently regulated and serve distinct functions in integrin signaling cascades. When designing experiments to study ITGB3 phosphorylation, researchers should carefully select antibodies specific to the phosphorylation site of interest and include appropriate controls to distinguish between these modifications.

What are the key signaling pathways involved in regulating ITGB3 Tyr773 phosphorylation?

ITGB3 Tyr773 phosphorylation is regulated by complex signaling networks:

Upstream Kinases:

• Focal Adhesion Kinase (FAK) has been implicated in ITGB3 phosphorylation and is required for endocytosis of extracellular vesicles

• Src family kinases can phosphorylate ITGB3 at tyrosine residues

Pathway Components:

• PTPRZ1-PTN Axis: Forms a complex with ITGB3 that stimulates endothelial cell migration through Tyr773 phosphorylation

• Ubiquitination Regulation: E3 ubiquitin ligases such as ITCH can modify ITGB3, potentially affecting its phosphorylation state

• ROS Signaling: Reactive oxygen species have been shown to induce ITGB3-mediated migration and invasion in colorectal cancer cells

Downstream Effectors:

• Phosphorylated ITGB3 at Tyr773 facilitates interactions with adaptor proteins

• Activates pathways involved in cell migration, proliferation, and invasion

• Mediates extracellular vesicle uptake through interactions with heparan sulfate proteoglycans (HSPGs)

Regulation Mechanisms:

• Ubiquitination by E3 ligases like ITCH can modify ITGB3 activity and potentially influence phosphorylation states

• Environmental factors such as oxidative stress can trigger ITGB3 phosphorylation

This complex network of interactions suggests that ITGB3 Tyr773 phosphorylation serves as an integration point for multiple signaling pathways, particularly those involved in cell migration and cancer metastasis.

How can I validate the specificity of Phospho-ITGB3 (Tyr773) antibodies in my experimental system?

Validating the specificity of Phospho-ITGB3 (Tyr773) antibodies is crucial for reliable experimental results. Consider implementing these validation approaches:

Site-Directed Mutagenesis Validation:

• Generate Y773F mutant ITGB3 constructs where tyrosine is replaced with phenylalanine

• Compare antibody reactivity between wild-type and Y773F mutant samples

• Absence of signal in the Y773F mutant confirms specificity

Phosphatase Treatment:

• Treat half of your sample with lambda phosphatase

• Compare antibody reactivity between treated and untreated samples

• Signal should disappear in phosphatase-treated samples

Peptide Competition Assay:

• Pre-incubate antibody with phospho-peptide containing the Tyr773 phosphorylation site

• Use non-phosphorylated peptide as a control

• Signal should be blocked by phospho-peptide but not by non-phospho-peptide

siRNA Knockdown:

• Transfect cells with siRNA targeting ITGB3 (e.g., 5′-CCGCTTCAATGAGGAAGTGAA-3′)

• Compare antibody reactivity in control versus ITGB3-depleted cells

• Signal should decrease in ITGB3-depleted samples

Immunoprecipitation-Western Blot Validation:

• Immunoprecipitate with anti-ITGB3 antibody

• Probe with anti-phosphotyrosine antibody

• Re-probe with Phospho-ITGB3 (Tyr773) specific antibody

• Signals should align, confirming specificity

Validation Data Example:
A validation experiment using melanoma cells transfected with wild-type or Y773F mutant human Integrin beta3 demonstrated the specificity of GTX25190 antibody. The antibody detected signal in phosphorylated wild-type ITGB3 but showed no reactivity with the Y773F mutant, confirming its specificity for the phosphorylated Tyr773 site .

What are the best practices for immunohistochemical detection of Phospho-ITGB3 (Tyr773) in tissue samples?

Tissue Preparation:

• Fix tissues in 4% paraformaldehyde

• Embed in paraffin or prepare frozen sections

• Cut sections at 4-6 μm thickness

Antigen Retrieval (Critical Step):

• Use heat-mediated antigen retrieval in EDTA buffer (pH 8.0)

• This step is essential for unmasking phospho-epitopes that may be obscured during fixation

Blocking Protocol:

• Block with 10% goat serum to reduce non-specific binding

• Incubate for 1 hour at room temperature

Antibody Incubation:

• Use primary antibody at 2 μg/ml concentration

• Incubate overnight at 4°C to maximize specific binding

• Use appropriate species-matched secondary antibody (e.g., Peroxidase Conjugated Goat Anti-rabbit IgG)

• Incubate secondary antibody for 30 minutes at 37°C

Detection System:

• For chromogenic detection, develop using DAB as the chromogen

• For fluorescent detection, use appropriate fluorophore-conjugated secondary antibodies

Controls to Include:

• Negative control: Omit primary antibody

• Phosphatase-treated section to demonstrate phospho-specificity

• Known positive tissue (e.g., human laryngeal squamous cell carcinoma, liver cancer, or thyroid cancer tissues have shown positive staining)

Optimization Tips:

• Titrate antibody concentration to determine optimal signal-to-noise ratio

• Adjust incubation times and temperatures as needed

• Consider using amplification systems for low-abundance phospho-proteins

Validated Tissue Types:
Phospho-ITGB3 (Tyr773) has been successfully detected in:

• Human laryngeal squamous cell carcinoma

• Human liver cancer tissue

• Human thyroid cancer tissue

• Rat lung tissue

What techniques can I use to study the functional significance of ITGB3 Tyr773 phosphorylation in cell migration and cancer metastasis?

To investigate the functional significance of ITGB3 Tyr773 phosphorylation in cell migration and cancer metastasis, consider these methodological approaches:

Genetic Manipulation:

• Site-Directed Mutagenesis:

  • Generate Y773F (non-phosphorylatable) and Y773E/D (phosphomimetic) ITGB3 mutants

  • Express in ITGB3-null or knockdown backgrounds

  • Compare phenotypes between wild-type and mutant expressing cells

• siRNA Knockdown and Rescue:

  • Deplete endogenous ITGB3 using siRNA (e.g., 5′-CCGCTTCAATGAGGAAGTGAA-3′)

  • Rescue with siRNA-resistant wild-type or Y773F mutant ITGB3

  • Assess recovery of cellular functions

Functional Assays:

• Cell Proliferation Assay:

  • Use Cell Counting Kit-8 (CCK-8) to measure proliferation rates

  • Measure at multiple timepoints (0, 12, 24, 48, and 72h)

  • Compare cells expressing wild-type vs. Y773F ITGB3

• Transwell Migration Assay:

  • Seed cells (3 × 10^5/mL) in serum-free medium in Matrigel-coated inserts

  • Add complete medium with 10% FBS to lower chamber

  • Incubate for 48 hours at 37°C

  • Quantify invading cells after fixation and crystal violet staining

• Extracellular Vesicle Uptake Assay:

  • Label EVs with fluorescent dyes

  • Measure uptake in cells expressing wild-type vs. Y773F ITGB3

  • Assess the role of FAK activation in this process

Molecular Interaction Studies:

• Co-Immunoprecipitation:

  • Immunoprecipitate with anti-ITGB3 or anti-ITCH antibodies

  • Detect interactions by Western blotting

  • Compare interactions between wild-type and Y773F mutant ITGB3

• Ubiquitination Analysis:

  • Immunoprecipitate ITGB3 and probe with anti-ubiquitin antibodies

  • Compare ubiquitination patterns between phosphorylated and non-phosphorylated ITGB3

In Vivo Models:

• Xenograft metastasis models comparing cells expressing wild-type vs. Y773F ITGB3

• Track metastatic spread using bioluminescence imaging

• Analyze tissue samples for ITGB3 Tyr773 phosphorylation status

This multifaceted approach will help establish the biological significance of ITGB3 Tyr773 phosphorylation in cellular functions related to cancer progression and metastasis.

How can I optimize immunofluorescence protocols for detecting Phospho-ITGB3 (Tyr773) in cultured cells?

Cell Preparation and Fixation:

• Grow cells on glass coverslips or chamber slides

• Fix with 4% paraformaldehyde for 15 minutes at room temperature

• Wash 3× with PBS

Permeabilization:

• Permeabilize with 0.1% Triton X-100 for 10 minutes at room temperature

• For membrane proteins like ITGB3, gentle permeabilization is crucial to preserve epitopes

Blocking:

• Block with 0.1-1% BSA in PBS for 30-60 minutes

• Add 5-10% normal serum from the secondary antibody host species to reduce background

Antibody Incubation:

• Dilute primary antibody to 1:250 in 0.1% BSA

• Incubate for 3 hours at room temperature or overnight at 4°C

• Wash 3× with PBS

• Incubate with fluorophore-conjugated secondary antibody (1:500-1:1000)

• Include nuclear counterstain (e.g., DAPI) and phalloidin for actin visualization if desired

Mounting and Imaging:

• Mount slides with anti-fade mounting medium

• Image using confocal or fluorescence microscopy

• Use appropriate filter sets for selected fluorophores

Controls and Validation:

• Include a no-primary antibody control to assess secondary antibody background

• Consider phosphatase treatment of some samples as a negative control

• Use known positive cell types (e.g., THP-1 cells have shown good detection)

Optimization Tips:

• Test different fixation methods if initial results are suboptimal

• Titrate antibody concentrations for optimal signal-to-noise ratio

• Consider signal amplification systems for low-abundance phospho-proteins

• To reduce background, extend washing steps and use 0.05% Tween-20 in wash buffer

Expected Results:
Successful immunofluorescence staining of Phospho-ITGB3 (Tyr773) typically shows membrane and/or cytoplasmic localization, with possible enrichment at focal adhesions or cell-cell junctions.

What are common issues with Phospho-ITGB3 (Tyr773) antibodies and how can they be resolved?

Problem 1: Weak or Absent Signal

Potential Causes:

• Low levels of ITGB3 Tyr773 phosphorylation in samples

• Phosphatase activity during sample preparation

• Epitope masking during fixation

• Insufficient antibody concentration

Solutions:

• Treat cells with phosphatase inhibitors during lysis (sodium orthovanadate, sodium fluoride)

• Optimize antigen retrieval for IHC/IF (EDTA buffer pH 8.0 is recommended)

• Increase antibody concentration or incubation time

• Stimulate cells to increase phosphorylation (e.g., with growth factors or integrin ligands)

Problem 2: High Background or Non-specific Staining

Potential Causes:

• Insufficient blocking

• Cross-reactivity with non-phosphorylated ITGB3 or related proteins

• Secondary antibody issues

• Overfixation of samples

Solutions:

• Increase blocking time or concentration (use 10% serum)

• Test different blocking agents (BSA, serum, commercial blockers)

• Pre-absorb antibody with non-phosphorylated peptide

• Reduce antibody concentration

• Include additional washing steps

Problem 3: Inconsistent Results Between Experiments

Potential Causes:

• Variability in phosphorylation status

• Differences in sample preparation

• Lot-to-lot antibody variation

• Cell culture conditions affecting baseline phosphorylation

Solutions:

• Standardize stimulation/treatment protocols

• Maintain consistent sample processing times

• Include positive controls in each experiment

• Consider using phosphatase inhibitors consistently

• Document antibody lot numbers and prepare large batches of working solutions

Problem 4: Unexpected Molecular Weight Bands in Western Blot

Potential Causes:

• Post-translational modifications altering mobility

• Proteolytic degradation

• Alternative splice variants

• Non-specific binding

Solutions:

• ITGB3 has an expected molecular weight of 87 kDa but often appears at 100-130 kDa due to glycosylation

• Include protease inhibitors during sample preparation

• Use fresh samples and avoid repeated freeze-thaw cycles

• Perform peptide competition assays to confirm specificity

• Compare with total ITGB3 antibody to confirm band identity

Problem 5: Poor Reproducibility in Phosphorylation Detection

Potential Causes:

• Rapid dephosphorylation during sample processing

• Variability in cell signaling states

• Inconsistent antibody performance

Solutions:

• Process samples rapidly and maintain at 4°C

• Use phosphatase inhibitor cocktails

• Standardize cell culture conditions (confluence, passage number)

• Consider using phosphomimetic mutants (Y773E/D) as positive controls

• Validate results with multiple detection methods

Consistent protocols and appropriate controls are key to reliable detection of Phospho-ITGB3 (Tyr773) across different experimental systems.

How does ITGB3 Tyr773 phosphorylation contribute to extracellular vesicle uptake and cancer metastasis?

Recent research has established a critical link between ITGB3 Tyr773 phosphorylation, extracellular vesicle (EV) uptake, and cancer metastasis, revealing a complex molecular mechanism:

Mechanism of EV Uptake:

• ITGB3 facilitates extracellular vesicle internalization through interactions with heparan sulfate proteoglycans (HSPGs)

• The process requires integrin endocytosis, which is influenced by Tyr773 phosphorylation

• Focal adhesion kinase (FAK) activation is needed for endocytosis of these vesicles

• This pathway represents a central mechanism in intercellular communication

Role in Cancer Metastasis:

• Metastasis accounts for 90% of cancer deaths and depends on tumor cell interactions with the microenvironment

• EVs secreted from primary tumors can modify both local and distant environments

• ITGB3-mediated uptake of EVs enables primary tumors to establish pre-metastatic niches

• Phosphorylation of ITGB3 at Tyr773 appears to be a regulatory point in this process

Experimental Evidence:

• Studies show ITGB3 is upregulated in ectopic endometrial stromal cells from patients with endometriosis

• ITCH-mediated ubiquitylation of ITGB3 promotes cell proliferation in these cells

• ITGB3 has been identified as a key regulator in reactive oxygen species-induced migration and invasion of colorectal cancer cells

• Knockout or functional blocking of ITGB3 significantly reduces EV uptake in experimental models

Potential Therapeutic Implications:

• Targeting ITGB3 Tyr773 phosphorylation could disrupt tumor-derived EV uptake

• This may provide a novel approach to preventing pre-metastatic niche formation

• Phospho-ITGB3 (Tyr773) antibodies could serve as research tools for developing targeted therapies

• Monitoring ITGB3 phosphorylation status might help assess metastatic potential

This emerging understanding of ITGB3 Tyr773 phosphorylation in EV-mediated communication offers new insights into cancer progression mechanisms and highlights potential intervention points for metastasis prevention.

What are the recommended controls when using Phospho-ITGB3 (Tyr773) antibodies in different applications?

Proper experimental controls are essential for validating results with Phospho-ITGB3 (Tyr773) antibodies. Here are application-specific control recommendations:

For Western Blotting:

Positive Controls:

• Cell lines known to express phosphorylated ITGB3 (e.g., HEL cells, human endothelial cells)

• Cells stimulated with growth factors or integrin ligands

• Recombinant phosphorylated ITGB3 peptide (if available)

Negative Controls:

• Phosphatase-treated samples

• Y773F mutant ITGB3 expressing cells

• ITGB3 knockdown/knockout samples

Specificity Controls:

• Peptide competition using phosphorylated and non-phosphorylated peptides

• Reprobing with total ITGB3 antibody to confirm protein identity

• Lane-to-lane loading controls (β-actin, GAPDH)

For Immunohistochemistry:

Positive Controls:

• Human laryngeal squamous cell carcinoma, liver cancer, or thyroid cancer tissues

• Rat lung tissue

• Tissues known to express phosphorylated ITGB3

Negative Controls:

• No primary antibody control

• Isotype control antibody

• Phosphatase-treated serial sections

• Non-expressing tissues

Validation Controls:

• Peptide competition

• Paired analysis with total ITGB3 staining on serial sections

For Immunofluorescence:

Essential Controls:

• No primary antibody control

• Untreated versus stimulated cells

• Y773F mutant ITGB3 expressing cells as negative control

Additional Controls:

• Dual staining with total ITGB3 to confirm localization

• ITGB3 knockdown cells

• Phosphatase-treated samples

For Immunoprecipitation:

Controls for Co-IP Experiments:

• Control IgG immunoprecipitation

• Reverse immunoprecipitation (pull down with interaction partner)

• Input lysate controls (5-10% of starting material)

• ITGB3 Y773F mutant comparison

For Functional Studies:

Genetic Controls:

• Wild-type ITGB3 expressing cells

• Y773F (non-phosphorylatable) ITGB3 expressing cells

• Y773E/D (phosphomimetic) ITGB3 expressing cells

• Vector-only transfected cells

Treatment Controls:

• Kinase inhibitors to prevent phosphorylation

• Phosphatase inhibitors to preserve phosphorylation

• Time course analysis to capture dynamic changes