Phospho-ITGB2 (T758) Antibody

What is Phospho-ITGB2 (T758) Antibody and what does it detect?

Phospho-ITGB2 (T758) Antibody is a specialized immunological reagent designed to detect integrin beta-2 (ITGB2, also known as CD18) protein specifically when phosphorylated at threonine residue 758. This antibody "detects endogenous levels of Integrin Beta 2 protein only when phosphorylated at T758" . Typically raised in rabbits, these antibodies are generated using synthetic peptides derived from human CD18/ITGB2 around the phosphorylation site, specifically within the amino acid range 720-769 . The high specificity for the phosphorylated form makes this antibody valuable for studying the activation state of ITGB2 in various cellular processes, particularly those related to immune cell function and signaling.

What are the common applications of Phospho-ITGB2 (T758) Antibody in research?

Phospho-ITGB2 (T758) Antibody serves multiple experimental purposes across different research methodologies:

• Western Blot (WB): For detecting phosphorylated ITGB2 in protein lysates, with recommended dilution ranges of 1:500-2000

• Immunohistochemistry (IHC-P): For visualizing phosphorylated ITGB2 in tissue sections, with recommended dilution ranges of 1:50-300

• Immunofluorescence (IF/ICC): For examining subcellular localization of phosphorylated ITGB2, typically used at dilutions of 1:200-1000

• ELISA: For quantitative detection of phosphorylated ITGB2 in experimental samples, used at much higher dilutions (1:40000)

These applications enable researchers to investigate ITGB2 phosphorylation status across various experimental systems, from cultured cells to patient-derived specimens.

What is the biological significance of ITGB2 phosphorylation at T758?

ITGB2 phosphorylation at T758 serves as a critical regulatory mechanism for integrin function. According to technical documentation, "Phosphorylation on Thr-758 (but not on Ser-756) allows interaction with 14-3-3 proteins" . This specific molecular interaction mediates several important biological processes:

• Regulation of leukocyte adhesion and transmigration, including T-cells and neutrophils

• Contribution to natural killer cell cytotoxicity in immune surveillance

• Involvement in neutrophil transmigration during inflammatory responses through PTK2B/PYK2-mediated activation

• Facilitation of apoptotic neutrophil phagocytosis by macrophages when ITGAL/ITGB2 associates with ICAM3

• Requirement for CD177-PRTN3-mediated activation of TNF-primed neutrophils (in association with alpha subunit ITGAM/CD11b)

The phosphorylation state therefore represents an activated form of the integrin that engages in specific downstream signaling pathways and functional outputs.

What recommended controls should be included when using Phospho-ITGB2 (T758) Antibody?

When designing experiments with Phospho-ITGB2 (T758) Antibody, several critical controls should be considered:

Positive controls:

• Cell populations known to express phosphorylated ITGB2 (e.g., activated leukocytes)

• Samples treated with agents known to induce ITGB2 phosphorylation (e.g., phorbol esters for T-cells)

Negative controls:

• Samples treated with phosphatase to remove phosphorylation

• Cell lines with ITGB2 knocked down or knocked out

• Secondary antibody-only controls to assess non-specific binding

Specificity controls:

• Competing peptide controls using the immunizing phosphopeptide

• Comparison with total ITGB2 antibody to assess phosphorylation relative to total protein levels

For disease-related studies such as cancer research, additional controls should include normal tissue counterparts and samples representing different disease stages to contextualize findings appropriately.

What are the optimal dilution ranges and conditions for different applications?

The recommended working dilutions for Phospho-ITGB2 (T758) Antibody vary by application:

For optimal storage, manufacturers recommend storing the antibody at -20°C for up to one year from receipt and avoiding repeated freeze-thaw cycles . The antibody is typically supplied in liquid form in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide as preservatives .

These parameters should be optimized for specific experimental conditions, considering factors such as target abundance, sample type, and detection method.

How can researchers validate the specificity of Phospho-ITGB2 (T758) Antibody?

Establishing antibody specificity is critical for reliable research outcomes. Several validation strategies include:

Peptide competition assays:

• Pre-incubate the antibody with the immunizing phosphopeptide before application

• A specific antibody will show reduced or eliminated signal when blocked with the specific phosphopeptide

Phosphatase treatment:

• Treat duplicate samples with lambda phosphatase to remove phosphorylation

• A phospho-specific antibody should show significantly reduced signal in treated samples

Genetic approaches:

• Compare cells expressing wild-type ITGB2 versus T758A mutants (preventing phosphorylation)

• The antibody should detect signal only in wild-type cells under phosphorylation-inducing conditions

Stimulation/inhibition experiments:

• Treat cells with stimuli known to induce ITGB2 phosphorylation (e.g., phorbol esters)

• Use inhibitors of relevant kinase pathways to prevent phosphorylation

• Monitor signal changes that should correlate with treatment conditions

These validation steps ensure that observed signals represent authentic phosphorylated ITGB2 rather than non-specific binding or cross-reactivity with similar epitopes.

What sample preparation techniques are recommended for preserving phosphorylation status?

Phosphorylation is a labile post-translational modification requiring special consideration during sample preparation:

Lysis conditions:

• Use buffers containing phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate)

• Include protease inhibitors to prevent protein degradation

• Maintain cold temperatures throughout processing

Cell/tissue handling:

• Minimize time between sample collection and processing

• Flash-freeze tissues in liquid nitrogen immediately after collection

• For adherent cells, consider direct lysis in culture dishes to minimize handling-induced changes

Fixation for microscopy:

• Use paraformaldehyde fixation (typically 4%) which better preserves phospho-epitopes

• Avoid prolonged fixation times that might mask epitopes

• Consider specialized phospho-epitope preservation fixatives for sensitive applications

Protein extraction for Western blotting:

• Use SDS-PAGE sample buffers containing phosphatase inhibitors

• Consider moderate heating of samples (65°C instead of 95°C) to minimize epitope disruption

• Process samples quickly and consistently across experimental groups

These considerations help maintain phosphorylation status throughout sample preparation, enabling accurate detection and quantification of the T758 phosphorylation site.

How can researchers optimize immunohistochemistry protocols for Phospho-ITGB2 detection?

Immunohistochemical detection of phosphorylated epitopes requires specific optimization:

Antigen retrieval:

• Test both heat-induced (HIER) and enzymatic retrieval methods

• For HIER, compare citrate buffer (pH 6.0) and EDTA buffer (pH 9.0)

• Optimize duration and temperature for maximum epitope exposure

Blocking and permeabilization:

• Use robust blocking (5-10% normal serum matching secondary antibody species)

• For intracellular epitopes, carefully titrate permeabilization reagents (0.1-0.5% Triton X-100)

• Consider specialized blocking reagents for phospho-epitopes to reduce background

Antibody concentration and incubation:

• Begin with manufacturer's recommended dilution range (1:50-300 for IHC-P)

• Test longer incubation times at 4°C versus shorter times at room temperature

• Consider signal amplification systems for low-abundance targets

Detection systems:

• Polymer-based detection systems often provide better signal-to-noise ratio

• Tyramide signal amplification can enhance sensitivity for rare epitopes

• Chromogen selection should consider counterstaining requirements and imaging method

Tissue-specific considerations:

• For tissues with high endogenous peroxidase, include additional quenching steps

• Consider section thickness (4-5μm typically optimal) and mounting substrates

• Fresh-cut sections often provide better results than stored slides

These optimization steps should be systematically documented to establish reproducible protocols for consistent results across experiments.

How does ITGB2 phosphorylation at T758 regulate protein-protein interactions?

ITGB2 phosphorylation at T758 creates a specific binding interface for regulatory proteins. According to technical data, "Phosphorylation on Thr-758 (but not on Ser-756) allows interaction with 14-3-3 proteins" . This molecular switch mechanism has several functional implications:

• 14-3-3 proteins function as molecular scaffolds connecting phosphorylated ITGB2 to downstream signaling components

• This interaction likely regulates ITGB2 conformation, affecting its binding affinity for extracellular ligands

• The binding may protect the phosphorylation site from dephosphorylation, prolonging the activated state

• 14-3-3 interaction potentially influences integrin clustering and lateral mobility in the membrane

This phosphorylation-dependent interaction represents a critical regulatory node in integrin-mediated signaling pathways and cellular functions.

What role does ITGB2 phosphorylation play in immune cell migration and adhesion?

ITGB2 phosphorylation at T758 serves as a crucial regulatory mechanism for immune cell dynamics:

• Regulates "leukocyte adhesion and transmigration of leukocytes including T-cells and neutrophils"

• "Triggers neutrophil transmigration during lung injury through PTK2B/PYK2-mediated activation"

• Modulates integrin affinity for ligands such as ICAM1, ICAM2, ICAM3, and ICAM4

• Contributes to "natural killer cell cytotoxicity" which requires appropriate cell-cell contact

The phosphorylation specifically influences these functions by:

• Altering ITGB2 conformation to modulate ligand binding properties

• Enabling recruitment of cytoskeletal and signaling proteins through 14-3-3 interaction

• Regulating integrin clustering and distribution in the membrane

• Potentially influencing recycling and endosomal trafficking of the integrin

These mechanisms collectively control the dynamic adhesion-de-adhesion cycles required for effective immune cell migration and function.

How can Phospho-ITGB2 (T758) Antibody be used to study immune cell functions?

The Phospho-ITGB2 (T758) Antibody provides a powerful tool for investigating immune cell dynamics:

Flow cytometry applications:

• Monitoring ITGB2 phosphorylation status during immune cell activation

• Correlating phosphorylation with expression of activation markers

• Identifying specific immune cell populations with active integrin signaling

Imaging applications:

• Visualizing the subcellular distribution of phosphorylated ITGB2 during immune synapse formation

• Tracking changes in phosphorylation during cell migration in real-time

• Co-localization studies with cytoskeletal components or signaling molecules

Functional correlation studies:

• Assessing ITGB2 phosphorylation in adhesion assays under static or flow conditions

• Correlating phosphorylation status with transmigration efficiency

• Examining phosphorylation changes in response to chemokine gradients

Cell-cell interaction studies:

• Investigating ITGB2 phosphorylation at immunological synapses

• Analyzing phosphorylation during leukocyte-endothelial interactions

• Studying phosphorylation dynamics during phagocytosis events

These approaches can provide mechanistic insights into how ITGB2 phosphorylation regulates diverse immune cell functions in health and disease.

What methods can be used to quantify ITGB2 phosphorylation in experimental samples?

Accurate quantification of ITGB2 phosphorylation requires appropriate methodological approaches:

Western blot-based quantification:

• Use Phospho-ITGB2 (T758) Antibody alongside total ITGB2 antibody

• Calculate phosphorylated/total ITGB2 ratio to normalize for expression differences

• Include loading controls (e.g., GAPDH, β-actin) for further normalization

• Employ digital imaging systems with sufficient dynamic range for accurate quantification

Flow cytometry-based quantification:

• Develop intracellular staining protocols using the Phospho-ITGB2 (T758) Antibody

• Measure median fluorescence intensity (MFI) to quantify phosphorylation levels

• Use appropriate controls including isotype and fluorescence-minus-one (FMO)

• Consider dual staining with total ITGB2 for calculating phosphorylation ratio

ELISA-based methods:

• Employ sandwich ELISA format using capture antibodies against total ITGB2

• Use Phospho-ITGB2 (T758) Antibody for detection at appropriate dilution (1:40000)

• Develop standard curves using recombinant phosphorylated protein

• Calculate phosphorylation levels relative to total protein concentration

Imaging-based quantification:

• Use standardized acquisition parameters for consistent measurement

• Apply automated image analysis algorithms to quantify signal intensity

• Normalize to cell number or area using appropriate counterstains

• Consider z-stack acquisition for three-dimensional analysis

These quantification approaches can be applied across different experimental systems to generate comparable data on ITGB2 phosphorylation dynamics.

How is ITGB2 expression and phosphorylation implicated in cancer progression?

Research on ITGB2 in cancer contexts has revealed significant correlations with disease progression:

• ITGB2 serves as "a potential marker for mesenchymal molecular subtype gliomas"

• "COX regression analysis shows that ITGB2 is an independent predictive marker of OS in malignant glioma patients"

• "Patients with high expression of ITGB2 in CGGA RNA-seq had lower OS in all grades of gliomas" with similar results obtained in "TCGA-GBMLGG RNA-seq set and Rembrandt Microarray data set"

• ITGB2 is involved in "glioma immune-related activities, especially closely related to B cells, CD4+Tcells, macrophages, neutrophils, and dendritic cells"

While the specific role of T758 phosphorylation in cancer contexts remains to be fully elucidated, the functional significance of this modification suggests it likely contributes to:

• Regulation of tumor-associated immune cell recruitment and function

• Modulation of the tumor microenvironment through altered immune cell interactions

• Potential influence on cancer cell migration and invasion through integrin-mediated processes

Research using Phospho-ITGB2 (T758) Antibody could help determine whether specific phosphorylation states correlate with tumor aggressiveness or treatment response.

What is the relationship between ITGB2 and immune cell infiltration in tumors?

Evidence indicates strong correlations between ITGB2 expression and immune infiltration in tumors:

• ITGB2 expression in Lower Grade Glioma (LGG) was "negatively correlated with tumor purity (r = 0.369, P < 0.05)" , indicating higher expression in samples with greater immune infiltration

• ITGB2 expression showed positive correlations with multiple immune cell populations:

  • B cells (r = 0.700, P < 0.05)

  • CD4+ T cells (r = 0.921, P < 0.05)

  • Macrophages (r = 0.820, P < 0.05)

  • Neutrophils (r = 0.836, P < 0.05)

  • Dendritic cells (r = 0.925, P < 0.05)

These strong correlations suggest ITGB2 plays a crucial role in immune cell recruitment and retention within tumors. The phosphorylation at T758 likely modulates these processes by:

• Regulating integrin activation state and ligand binding affinity

• Influencing immune cell adhesion to endothelium during extravasation

• Mediating interactions with extracellular matrix components within the tumor microenvironment

• Participating in immune cell activation through outside-in signaling

These findings highlight the potential importance of ITGB2 phosphorylation in tumor immunology and immunotherapy response.

How does ITGB2 methylation relate to its expression and phosphorylation in cancer?

Research has revealed an interesting relationship between ITGB2 methylation and expression in cancer:

• "ITGB2 is negatively regulated by ITGB2 methylation, resulting in low expression in LGG tissues"

• "Low expression of ITGB2 and high methylation indicate good OS in patients with LGG"

• An "ITGB2 methylation risk score (ITMRS) obtained from the ITGB2 methylation CpG site can better predict the OS of LGG patients"

• Methylation-dependent suppression of ITGB2 expression reducing the substrate available for phosphorylation

• Potential coordinated regulation of both modifications in specific cancer subtypes

• The possibility that methylation status influences the molecular context in which phosphorylation occurs

These findings highlight the complexity of ITGB2 regulation in cancer and suggest that integrating methylation and phosphorylation data could provide more comprehensive insights into its role in disease progression.

What methodological approaches are recommended for studying ITGB2 phosphorylation in patient-derived samples?

Investigating ITGB2 phosphorylation in clinical specimens requires specialized approaches:

Sample collection and preservation:

• Rapid processing with phosphatase inhibitors immediately after procurement

• Standardized fixation protocols optimized for phospho-epitope preservation

• Consider parallel sampling for multiple analytical approaches

Multiplexed tissue analysis:

• Implement multiplexed immunohistochemistry/immunofluorescence with Phospho-ITGB2 (T758) Antibody

• Include markers for total ITGB2, cell lineage identification, and other signaling molecules

• Use multispectral imaging for comprehensive analysis of complex tissue environments

Single-cell approaches:

• Employ gentle tissue disaggregation with phosphatase inhibitors

• Implement multiparameter flow cytometry with Phospho-ITGB2 (T758) Antibody

• Consider single-cell phosphoproteomics for comprehensive phosphorylation profiling

Digital pathology integration:

• Develop quantitative image analysis workflows for phospho-ITGB2 staining

• Implement machine learning approaches for pattern recognition

• Correlate phospho-ITGB2 spatial distribution with histopathological features

Clinical correlation analyses:

• Correlate phospho-ITGB2 patterns with patient outcomes, treatment responses

• Perform multivariate analyses incorporating other molecular and clinical parameters

• Consider temporal analyses using longitudinal samples when available

These methodological approaches can help translate basic research findings into clinically relevant insights about ITGB2 phosphorylation in disease contexts.