ITGB1 (Ab-789) Antibody

What is ITGB1 and why is it significant in cellular biology?

ITGB1 (Integrin beta 1) is a critical transmembrane receptor subunit that forms heterodimeric complexes with various alpha integrin subunits. These heterodimers function as primary receptors for extracellular matrix proteins including collagen, fibronectin, laminin, and vitronectin. ITGB1 plays fundamental roles in cell adhesion and recognition across numerous biological processes including embryogenesis, hemostasis, tissue repair, immune response, and tumor cell metastasis . The significance of ITGB1 extends to its ability to recognize the RGD sequence in multiple ligands and its involvement in promoting endothelial cell motility, angiogenesis, and osteoblast compaction through fibronectin fibrillogenesis . Additionally, ITGB1 has emerged as a key player in viral infection processes, particularly in rabies virus entry mechanisms, making it an important target for both cellular biology and virology research .

What does the phosphorylation at threonine 789 indicate about ITGB1 function?

Phosphorylation at threonine 789 (T789) represents a crucial post-translational modification that regulates ITGB1 functionality. The ITGB1 (Ab-789) antibody specifically recognizes the phosphorylated form at this residue, which is associated with altered integrin signaling and trafficking . This phosphorylation event affects the protein's interaction capabilities with cytoskeletal components and downstream signaling molecules, influencing cellular processes such as adhesion, migration, and proliferation. Research indicates that phosphorylation at T789 may play a regulatory role in the binding affinity of ITGB1 for its ligands and partners, potentially modulating its involvement in processes like viral entry, as seen with rabies virus infection mechanisms . The ability to specifically detect this phosphorylated form allows researchers to investigate how signaling pathways influence integrin-mediated cellular responses.

What applications is the ITGB1 (Ab-789) antibody validated for?

The ITGB1 (Ab-789) antibody has been validated for multiple research applications that enable comprehensive investigation of this protein's expression and function. According to the product specifications, this rabbit polyclonal antibody is suitable for:

• Western Blot (WB) analysis with recommended dilutions of 1:500-1:1000

• Immunohistochemistry (IHC) with paraffin-fixed tissues at dilutions of 1:50-1:100

• Immunocytochemistry (ICC)

These applications allow researchers to detect and quantify phosphorylated ITGB1 in various experimental setups, from protein expression analysis to cellular localization studies. The antibody has been cited in multiple publications, indicating its reliability and acceptance in the scientific community . When designing experiments, researchers should note that optimization of working dilutions may be necessary depending on the specific experimental conditions and sample types.

How can ITGB1 (Ab-789) antibody be used to investigate viral entry mechanisms?

The ITGB1 (Ab-789) antibody serves as a valuable tool for investigating viral entry mechanisms, particularly for viruses that utilize integrins as entry factors. Research has demonstrated that ITGB1 plays a crucial role in rabies virus (RABV) peripheral entry, making this antibody especially useful for studying virus-host interactions .

To effectively investigate viral entry using this antibody, researchers can employ several methodological approaches:

• Antibody blocking assays: Pre-treat cells with the ITGB1 antibody before viral infection to assess its ability to block viral entry. Studies have shown that ITGB1-specific antibodies can significantly decrease viral infection, as demonstrated with RABV .

• Co-immunoprecipitation studies: Use the antibody to examine direct interactions between ITGB1 and viral proteins. The search results indicate successful co-IP assays between ITGB1 and RABV G protein, confirming their direct interaction .

• Confocal microscopy with co-localization analysis: Utilize the antibody to track the internalization and intracellular trafficking of ITGB1 alongside viral particles. Research has shown that ITGB1 is internalized into cells and transported to late endosomes together with RABV .

• Phosphorylation state analysis: Investigate how viral infection affects the phosphorylation status of ITGB1 at T789, potentially revealing regulatory mechanisms of virus-induced signaling.

These approaches can provide valuable insights into how viruses exploit ITGB1 for cellular entry and how phosphorylation at T789 might regulate this process.

What experimental controls should be implemented when using the ITGB1 (Ab-789) antibody?

Implementing appropriate controls is essential for generating reliable data with the ITGB1 (Ab-789) antibody. Researchers should consider the following control strategies:

• Phosphorylation-specific validation: Since this antibody detects ITGB1 only when phosphorylated at threonine 789, include samples treated with phosphatase to confirm specificity . This control verifies that the signal truly represents the phosphorylated form.

• Peptide competition assay: Pre-incubate the antibody with the immunizing phosphopeptide to block specific binding sites. The antibody was raised against a synthetic phosphopeptide corresponding to residues surrounding T789 of human ITGB1 , making this an appropriate specificity control.

• Knockdown/knockout controls: Include ITGB1 siRNA-treated samples as negative controls. Research has demonstrated that siRNA targeting human or mouse ITGB1 mRNA reduced expression by 62% and 48%, respectively .

• Overexpression controls: Include samples overexpressing ITGB1 as positive controls. Studies have shown that transient transfection with ITGB1 cDNA (p-ITGB1) increases detectable levels .

• Cross-reactivity assessment: When working with different species, validate antibody specificity across species boundaries. The antibody has been reported to react with human, mouse, and rat samples .

Implementation of these controls ensures experimental rigor and supports the validity of research findings involving ITGB1 phosphorylation states.

How does ITGB1 interact with fibronectin and how can this be studied using the antibody?

ITGB1 interaction with fibronectin (FN) represents a critical aspect of integrin function that can be effectively studied using the ITGB1 (Ab-789) antibody. This interaction occurs through the recognition of RGD (Arg-Gly-Asp) motifs in fibronectin by ITGB1-containing integrin heterodimers .

Methodological approaches to study this interaction include:

• Co-immunoprecipitation assays: Research has successfully demonstrated that Flag-tagged FN (FN-Flag) interacts with Myc-tagged ITGB1 (ITGB1-Myc) in plasmid-coexpressed cell lysates . The ITGB1 (Ab-789) antibody can be employed to detect the phosphorylated form of ITGB1 in these complexes.

• RGD peptide competition assays: Studies have shown that RGD peptides can block RABV infection in both cell culture and animal models, indicating the importance of the ITGB1-fibronectin interaction . Researchers can use these peptides as competitive inhibitors to assess the functional significance of phosphorylation at T789.

• Phosphorylation-dependent binding analysis: The antibody can help determine whether phosphorylation at T789 affects the binding affinity of ITGB1 for fibronectin by comparing immunoprecipitation efficiency between phosphorylated and non-phosphorylated forms.

• Immunofluorescence co-localization: Co-staining for phosphorylated ITGB1 and fibronectin can reveal spatial relationships at cellular adhesion sites and how phosphorylation might regulate these interactions.

This methodological toolkit allows researchers to dissect the complex interplay between ITGB1 phosphorylation states and fibronectin binding, potentially revealing regulatory mechanisms in cell adhesion, migration, and viral infection processes.

What are common issues when using ITGB1 (Ab-789) antibody in Western blotting and how can they be resolved?

When utilizing the ITGB1 (Ab-789) antibody for Western blotting, researchers may encounter several challenges that require specific optimization strategies:

• High background signal: This common issue can be addressed by:

  • Increasing blocking time and concentration (5% BSA is often more effective than milk for phospho-specific antibodies)

  • Using more stringent washing conditions (increasing TBST concentration or wash duration)

  • Diluting the antibody further (testing the upper end of the recommended 1:500-1:1000 range)

  • Including phosphatase inhibitors in all sample preparation buffers to preserve the phosphorylation state

• Weak or absent signal: To enhance detection of phosphorylated ITGB1:

  • Enrich for membrane proteins during sample preparation

  • Use phosphatase inhibitor cocktails during cell lysis

  • Consider stimulating cells with growth factors or adhesion to fibronectin to increase T789 phosphorylation

  • Optimize protein loading (50-100 μg of total protein is typically recommended)

  • Reduce the antibody dilution within the recommended range

• Multiple bands or unexpected molecular weight: ITGB1 undergoes extensive post-translational modifications including glycosylation. Researchers should:

  • Use proper molecular weight markers (ITGB1 runs at approximately 130 kDa when fully processed)

  • Consider running reduced and non-reduced samples in parallel

  • Include positive control lysates from cells known to express phosphorylated ITGB1

Implementation of these strategies should optimize detection of phosphorylated ITGB1 in Western blotting applications, leading to more reliable and interpretable results.

How can researchers optimize immunohistochemistry protocols for ITGB1 (Ab-789) antibody?

Optimizing immunohistochemistry protocols for the ITGB1 (Ab-789) antibody requires careful attention to several parameters:

• Antigen retrieval optimization:

  • Test both heat-induced epitope retrieval (HIER) methods with citrate buffer (pH 6.0) and EDTA buffer (pH 9.0)

  • Optimize retrieval time (typically 15-20 minutes)

  • For phospho-epitopes, EDTA-based buffers often provide superior results

• Antibody incubation conditions:

  • Start with the manufacturer's recommended dilution range (1:50-1:100)

  • Test both room temperature (1-2 hours) and 4°C overnight incubation

  • Consider using a humidity chamber to prevent tissue drying

• Signal amplification and detection systems:

  • For phospho-specific antibodies, tyramide signal amplification systems may improve sensitivity

  • Compare DAB and AEC chromogens for optimal signal-to-noise ratio

  • Consider fluorescent secondary antibodies for co-localization studies

• Tissue-specific considerations:

  • Use PFA-fixed tissues as recommended in the specifications

  • Include positive control tissues known to express phosphorylated ITGB1

  • For muscle tissue analysis (relevant for RABV studies), perfusion fixation may improve antigen preservation

• Phosphorylation preservation:

  • Minimize time between tissue collection and fixation

  • Include phosphatase inhibitors in fixatives when possible

  • Consider phosphatase treatment of control sections to verify phospho-specificity

By systematically optimizing these parameters, researchers can achieve consistent and specific staining of phosphorylated ITGB1 in tissue sections, enabling reliable analysis of its expression and localization in various physiological and pathological contexts.

How can ITGB1 (Ab-789) antibody be used to investigate the role of ITGB1 in RABV infection?

The ITGB1 (Ab-789) antibody presents a valuable tool for investigating the specific role of phosphorylated ITGB1 in RABV infection through several sophisticated experimental approaches:

• Infection inhibition assays: Research has demonstrated that antibodies against ITGB1 can significantly block cell-adapted RABV infection in cells and street RABV infection in mice via intramuscular (but not intracerebral) inoculation . Researchers can use the phospho-specific antibody to determine if the phosphorylated form is specifically involved in this process.

• Phosphorylation dynamics during infection: Using the antibody, researchers can track changes in ITGB1 T789 phosphorylation status at different time points during RABV infection to establish temporal relationships between phosphorylation events and viral entry or replication.

• Co-localization with viral components: Immunofluorescence studies using the antibody can reveal:

  • Whether phosphorylated ITGB1 specifically co-localizes with viral particles during entry

  • If phosphorylated ITGB1 is enriched at sites of virus attachment

  • How phosphorylation affects trafficking to late endosomes where RABV has been shown to co-transport with ITGB1

• Correlation with nAChRα1 interaction: Since ITGB1 interacts with nicotinic acetylcholine receptor (nAChRα1), which is a proposed receptor for peripheral RABV infection , the antibody can help determine if phosphorylation at T789 affects this interaction and subsequent viral entry.

• In vivo localization studies: For animal models, immunohistochemistry using this antibody can reveal the distribution of phosphorylated ITGB1 at RABV inoculation sites in mouse muscle tissue, potentially identifying critical cellular populations involved in initial viral entry.

These approaches can significantly advance our understanding of how ITGB1 phosphorylation states influence viral infection mechanisms, potentially revealing novel therapeutic targets.

What methodological approaches can be used to study the interaction between ITGB1 and viral glycoproteins?

Investigating the interaction between ITGB1 and viral glycoproteins, such as RABV G, requires sophisticated methodological approaches where the ITGB1 (Ab-789) antibody can play a pivotal role:

• Biochemical interaction assays:

  • Co-immunoprecipitation (Co-IP): Research has successfully demonstrated that Flag-tagged ITGB1 interacts with Myc-tagged RABV G in HEK293 cell lysates . Researchers can use the phospho-specific antibody to determine if T789 phosphorylation affects this interaction.

  • GST pulldown assays: Studies have shown that GST-tagged ITGB1 ectodomain (ITGB1 ED) can pull down N-terminal His-tagged RABV G ectodomain (ERAG ED) , confirming direct interaction. The influence of phosphorylation status on this interaction can be investigated using phospho-mimetic mutations.

• Biophysical interaction analysis:

  • Surface Plasmon Resonance (SPR): Measure binding kinetics between purified ITGB1 and viral glycoproteins, comparing phosphorylated and non-phosphorylated forms

  • Microscale Thermophoresis (MST): Assess binding affinities under various conditions to determine how phosphorylation affects interaction strength

• Structural studies:

  • Cryo-EM analysis: Investigate structural arrangements of ITGB1-viral glycoprotein complexes

  • Hydrogen-deuterium exchange mass spectrometry: Identify the specific binding interfaces involved in the interaction

• Cell-based assays:

  • FRET/BRET analysis: Measure real-time interactions in living cells

  • Proximity ligation assay (PLA): Visualize interactions within cells with high sensitivity and spatial resolution

  • Live-cell imaging: Track the dynamics of fluorescently labeled ITGB1 and viral glycoproteins during viral entry

• Computational approaches:

  • Molecular docking: Predict interaction interfaces between ITGB1 and viral glycoproteins

  • Molecular dynamics simulations: Assess how phosphorylation might alter binding properties

These methodological approaches provide a comprehensive toolkit for investigating the molecular interactions between ITGB1 and viral glycoproteins, with particular emphasis on how phosphorylation at T789 might regulate these interactions.

How does ITGB1 knockdown/overexpression affect viral infection and how can this be quantified?

Research has demonstrated that modulation of ITGB1 expression significantly impacts viral infection rates, particularly for rabies virus. Implementing proper quantification methods with the ITGB1 (Ab-789) antibody can provide valuable insights into these effects:

• Quantitative analysis of ITGB1 knockdown effects:
  Research has shown that siRNA-mediated silencing of ITGB1 drastically reduces RABV infection:

  Cell Type	ITGB1 Reduction	Relative Infection Rate Decrease	RABV Growth Titer Effect
  HEK293	62%	72%	Significantly lower
  N2a	48%	87%	Significantly lower

  These findings demonstrate that even partial reduction of ITGB1 expression can lead to substantial decreases in viral infection rates .

• Quantification of overexpression effects:

  • Transient transfection with ITGB1 cDNA has been shown to moderately increase RABV growth titers at 24, 36, and 48 hours post-infection

  • The ITGB1 (Ab-789) antibody can help determine if increased phosphorylation at T789 correlates with enhanced viral replication

• Methodological approaches for quantification:

  • Flow cytometry: Measure infection rates in populations with varying ITGB1 expression levels

  • Plaque assays: Quantify infectious viral particle production

  • qRT-PCR: Assess viral RNA replication

  • Western blotting: Determine viral protein expression levels alongside ITGB1 phosphorylation status

• Time-course analysis:

  • Track phosphorylation changes at T789 throughout the viral replication cycle

  • Correlate phosphorylation status with specific stages of viral entry and replication

• Pathway inhibition studies:

  • Use kinase or phosphatase inhibitors to modulate ITGB1 phosphorylation

  • Assess effects on viral entry and replication

These quantification methods provide comprehensive approaches to understand how ITGB1 expression levels and phosphorylation status influence viral infection processes, potentially revealing therapeutic targets for antiviral development.

How does the neutralization capacity of ITGB1 ectodomain compare to antibody-mediated blocking in viral infection studies?

Research comparing ITGB1 ectodomain soluble protein with antibody-mediated blocking offers valuable insights into intervention strategies against ITGB1-dependent viral infections:

• ITGB1 ectodomain neutralization efficacy:
  Studies have demonstrated dose-dependent neutralization of RABV by ITGB1 ectodomain (ITGB1 ED):

  Cell Type	ITGB1 ED Concentration	Relative Infection Rate Reduction	Cell Viability Effect
  HEK293	25 μg/ml	35%	No effect
  HEK293	100 μg/ml	98%	No effect
  N2a	25 μg/ml	18%	No effect
  N2a	100 μg/ml	92%	No effect

  These results indicate high neutralization efficacy at higher concentrations without cytotoxicity .

• Antibody-mediated blocking:

  • ITGB1-specific antibodies have been shown to block viral infection in various experimental settings

  • The phospho-specific ITGB1 (Ab-789) antibody can help determine if targeting the phosphorylated form provides enhanced or selective inhibition

• Comparative analysis methods:

  • Dose-response curves: Compare EC50 values between ectodomain and antibody approaches

  • Infection timing studies: Determine at which stages of infection each approach is most effective

  • Combination treatments: Assess potential synergistic effects

  • In vivo validation: Compare efficacy in animal models via different administration routes

• Mechanistic distinctions:

  • Ectodomain likely functions through competitive inhibition of virus-receptor binding

  • Antibodies may induce additional effects including receptor clustering, internalization, or conformational changes

  • The ITGB1 (Ab-789) antibody can reveal if phosphorylation state affects these mechanisms

• Therapeutic implications:

  • The significant differences in neutralization between intramuscular and intracerebral routes suggest tissue-specific mechanisms

  • Understanding phosphorylation-dependent effects may inform targeted therapeutic approaches

This comparative analysis helps researchers evaluate different strategies for targeting ITGB1-dependent viral entry, potentially informing the development of postexposure treatments for rabies and other viruses that utilize similar entry mechanisms.

How might the phosphorylation state of ITGB1 at T789 regulate its role in viral entry mechanisms?

Understanding how phosphorylation at threonine 789 affects ITGB1's function in viral entry presents an exciting frontier for future research. The ITGB1 (Ab-789) antibody provides a valuable tool for investigating several key questions:

• Phosphorylation-dependent conformational changes:

  • Does T789 phosphorylation alter the three-dimensional structure of ITGB1, potentially exposing or concealing viral binding sites?

  • How does phosphorylation affect the activation state of integrin heterodimers that contain ITGB1?

  • Can structural analysis using the antibody reveal phosphorylation-induced conformational changes?

• Signaling pathway modulation:

  • What kinases and phosphatases regulate T789 phosphorylation during viral infection?

  • Does viral binding to ITGB1 trigger changes in its phosphorylation status?

  • How does T789 phosphorylation affect downstream signaling cascades that might facilitate viral entry?

• Trafficking and internalization dynamics:

  • Does phosphorylation at T789 influence the rate or route of ITGB1 internalization during viral entry?

  • How does phosphorylation affect sorting and trafficking to specific endosomal compartments where viral fusion might occur?

  • Does phosphorylation status impact recycling rates of ITGB1 to the cell surface?

• Interaction with co-receptors:

  • How does T789 phosphorylation influence ITGB1's interaction with nAChRα1, which research has identified as a key factor in RABV peripheral infection ?

  • Are there phosphorylation-dependent changes in ITGB1's interaction with other potential co-receptors or entry factors?

• Therapeutic targeting approaches:

  • Could small molecules that modulate T789 phosphorylation serve as antiviral agents?

  • Might phosphorylation-specific antibodies like ITGB1 (Ab-789) themselves have therapeutic potential?

These research directions could significantly advance our understanding of how post-translational modifications regulate virus-host interactions, potentially revealing novel intervention strategies for ITGB1-dependent viral infections.

What experimental designs would best evaluate ITGB1 as a therapeutic target for rabies and other viral infections?

Developing experimental designs to evaluate ITGB1 as a therapeutic target requires comprehensive approaches that address both efficacy and mechanism of action:

• In vitro screening platforms:

  • High-throughput screening: Develop assays to identify compounds that modulate ITGB1 phosphorylation at T789

  • Time-of-addition studies: Determine the temporal window during which ITGB1-targeting is effective

  • Cell type specificity analysis: Assess efficacy across relevant cell types (neurons, muscle cells) using the ITGB1 (Ab-789) antibody to monitor phosphorylation status

• Animal model evaluation:

  • Post-exposure prophylaxis model: Test ITGB1-targeting approaches as post-exposure treatments for RABV

  • Route-comparative studies: Compare efficacy via different administration routes, given the observation that ITGB1-blocking affects intramuscular but not intracerebral RABV infection

  • Combination therapy assessment: Evaluate synergy with existing rabies immunoglobulin treatments

• Mechanism characterization studies:

  • Structure-function analysis: Identify the minimal ITGB1 domains required for viral binding

  • Mutational studies: Generate phosphomimetic (T789D/E) and phosphodeficient (T789A) mutants to assess functional consequences

  • Conformational antibody panels: Develop antibodies recognizing different conformational states of ITGB1

• Therapeutic agent development:

  • Soluble receptor mimetics: Optimize ITGB1 ectodomain designs based on neutralization efficacy data

  • Antibody engineering: Develop antibodies specifically targeting the viral-binding interface

  • Small molecule inhibitors: Screen for compounds that disrupt ITGB1-viral glycoprotein interactions

• Translational considerations:

  • Biodistribution studies: Track labeled therapeutic agents to ensure they reach relevant tissues

  • Safety profiles: Assess potential side effects of ITGB1 targeting on integrin-dependent physiological processes

  • Stability and formulation: Optimize therapeutic agent stability for field applications

These experimental approaches provide a comprehensive framework for evaluating ITGB1 as a therapeutic target, with particular emphasis on how phosphorylation status might influence efficacy and specificity of intervention strategies.