Phospho-ICAM1 (Tyr512) Antibody

What is the Phospho-ICAM1 (Tyr512) Antibody and what specifically does it detect?

The Phospho-ICAM1 (Tyr512) Antibody is a specialized immunological reagent that selectively detects endogenous levels of Intercellular Adhesion Molecule 1 (ICAM1) protein only when it is phosphorylated at the Tyrosine 512 residue. This antibody cannot recognize the non-phosphorylated form of ICAM1 or ICAM1 phosphorylated at different sites, making it a highly specific tool for studying this particular post-translational modification. The antibody is typically produced in rabbits as a polyclonal IgG using synthetic peptides derived from human ICAM1 around the phosphorylation site of Tyr512, usually corresponding to amino acids 479-528 of the protein sequence.

How is the specificity of Phospho-ICAM1 (Tyr512) Antibody validated in research settings?

The specificity of Phospho-ICAM1 (Tyr512) Antibody is validated through multiple complementary approaches. A superior validation strategy involves the use of blocking peptides, which can block the signal of the antibody in a Western Blotting assay, ensuring site-specificity. This validation confirms that the antibody recognizes only the phosphorylated target protein at Tyr512, not the non-phosphorylated ICAM1 or ICAM1 phosphorylated at different sites. Additionally, researchers may employ phosphomimetic mutants (where Tyr512 is substituted with aspartic acid) and phospho-null mutants (where Tyr512 is substituted with alanine) to further confirm specificity in cellular systems.

What is the biological significance of ICAM1 phosphorylation at Tyr512?

Phosphorylation of ICAM1 at Tyr512 plays a crucial role in regulating protein-protein interactions and downstream signaling pathways. Recent research has revealed that the Tyr512 residue of ICAM1 can directly bind to SRC, thereby regulating SRC signaling activity. This phosphorylation is mediated by tyrosine-protein kinase Met (c-MET), and the phosphorylated form of ICAM1 interacts with SRC to increase its activity. This molecular interaction has significant implications in cancer progression, particularly through mechanisms involving epithelial-mesenchymal transition (EMT) and angiogenesis. The phosphorylation status at this specific residue represents a critical regulatory mechanism in ICAM1-mediated cellular functions.

What are the recommended applications and optimal dilution ranges for Phospho-ICAM1 (Tyr512) Antibody?

The Phospho-ICAM1 (Tyr512) Antibody has been validated for several experimental applications with specific optimal dilution ranges:

These dilution guidelines should be considered starting points that may require optimization based on your specific experimental conditions, sample type, and detection method. When establishing a new protocol, it is advisable to test a range of dilutions to determine the optimal antibody concentration for your particular application.

How should Phospho-ICAM1 (Tyr512) Antibody be stored to maintain its activity?

For optimal maintenance of antibody activity, Phospho-ICAM1 (Tyr512) Antibody should be stored at -20°C for long-term storage. The antibody is typically supplied in phosphate buffered saline (pH 7.4, 150mM NaCl) containing 50% glycerol and 0.02% sodium azide as preservatives. Under these storage conditions, the antibody remains stable for up to 12 months from the date of receipt. For shorter-term storage (up to 6 months), the antibody can be kept at 4°C. It is crucial to avoid repeated freeze-thaw cycles, as these can significantly compromise antibody activity and specificity. Aliquoting the antibody upon receipt is recommended if multiple uses are anticipated over an extended period.

What controls should be included when using Phospho-ICAM1 (Tyr512) Antibody in Western blotting experiments?

A robust Western blotting experiment using Phospho-ICAM1 (Tyr512) Antibody should include the following controls:

• Positive Control: Lysates from cells known to express phosphorylated ICAM1 at Tyr512, such as cytokine-stimulated endothelial cells or certain cancer cell lines (e.g., SW-480 colorectal cancer cells).

• Negative Control: Lysates from cells treated with phosphatase to remove phosphorylation, or cells where ICAM1 expression has been knocked down using siRNA or CRISPR-Cas9.

• Phosphorylation-Specific Controls:

  • Samples treated with c-MET inhibitors to prevent phosphorylation of ICAM1 at Tyr512

  • Samples treated with HGF (hepatocyte growth factor, a c-MET ligand) to enhance phosphorylation

• Blocking Peptide Control: Running parallel blots with the antibody pre-incubated with its specific blocking peptide to demonstrate specificity.

• Loading Control: An antibody against a housekeeping protein (e.g., GAPDH, β-actin) to ensure equal loading across lanes.

Including these controls helps validate antibody specificity and ensures accurate interpretation of experimental results, particularly when examining changes in phosphorylation status under different experimental conditions.

How does ICAM1 phosphorylation at Tyr512 contribute to cancer progression?

ICAM1 phosphorylation at Tyr512 plays a significant role in cancer progression through several mechanisms:

• SRC Signaling Activation: Phosphorylated ICAM1 (Tyr512) directly binds to SRC, increasing its kinase activity. This enhanced SRC activity promotes cancer cell migration, invasion, and metastasis.

• Regulation of EMT: Through SRC activation, phosphorylated ICAM1 upregulates EMT markers including N-cadherin, Vimentin, Snail, and Slug, facilitating cancer cell transition to a more invasive phenotype.

• Promotion of Angiogenesis: ICAM1-SRC signaling increases the expression of angiogenic factors such as VEGF-A and PDGF-BB, enhancing tumor vascularization.

• Positive Feedback Loop: A positive feedback mechanism involving ICAM1/SRC/STAT3 further amplifies these oncogenic signals, as revealed by decreased ICAM1 expression when c-MET inhibitors are administered.

Analysis of Cancer Genome Atlas (TCGA) data has demonstrated a positive correlation between ICAM1 expression and phosphorylated SRC levels in colorectal cancer patients. Gene Set Enrichment Analysis (GSEA) further confirms a positive correlation between ICAM1 and the SRC oncogenic signature, highlighting the clinical relevance of this signaling axis in cancer biology.

What experimental approaches can be used to study the role of phosphorylated ICAM1 (Tyr512) in disease models?

Several experimental approaches can be employed to investigate the role of phosphorylated ICAM1 (Tyr512) in disease models:

• Phosphomimetic and Phospho-null Mutants: Generate ICAM1 constructs where Tyr512 is replaced with either aspartic acid (phosphomimetic) or alanine (phospho-null) to study the functional consequences of phosphorylation in vitro and in vivo.

• Kinase Inhibition Studies: Utilize c-MET inhibitors to prevent ICAM1 phosphorylation and examine the effects on downstream signaling, cell migration, invasion, and angiogenesis.

• Neutralizing Antibody Treatments: Apply ICAM1 neutralizing antibodies to block its function and assess the impact on SRC activity and cancer-related processes, both in vitro and in xenograft models.

• Co-immunoprecipitation (Co-IP) and Proximity Ligation Assay (PLA): Employ these techniques to study the physical interaction between phosphorylated ICAM1 and SRC under various experimental conditions.

• In Vivo Models: Develop xenograft models using cells expressing wild-type, phosphomimetic, or phospho-null ICAM1 to evaluate tumor growth, metastasis, and response to targeted therapies.

• Patient-Derived Samples: Analyze clinical specimens for correlations between ICAM1 phosphorylation status, SRC activity, and clinical outcomes using the Phospho-ICAM1 (Tyr512) Antibody for immunohistochemistry.

What is the molecular mechanism by which c-MET regulates ICAM1 phosphorylation at Tyr512?

The molecular mechanism of c-MET-mediated ICAM1 phosphorylation involves a sophisticated signaling cascade:

• Direct Phosphorylation: c-MET directly phosphorylates ICAM1 at Tyr512, as demonstrated by c-MET kinase assays using purified ICAM1 protein. This direct kinase-substrate relationship is critical for initiating downstream signaling.

• HGF-Mediated Activation: Hepatocyte growth factor (HGF), the ligand for c-MET, enhances this phosphorylation. Treatment of cells with HGF increases phosphorylated SRC levels, but this effect is diminished when ICAM1 expression is silenced, indicating that ICAM1 is an essential intermediate in HGF/c-MET/SRC signaling.

• Synergistic Effect: Co-expression of ICAM1 and c-MET significantly amplifies SRC activity beyond the effect of either protein alone, suggesting a cooperative mechanism in signal transduction.

• Feedback Regulation: A positive feedback loop exists wherein c-MET-phosphorylated ICAM1 activates SRC, which subsequently enhances STAT3 signaling, further increasing ICAM1 expression. This is evidenced by decreased total ICAM1 expression upon c-MET inhibitor treatment.

This intricate signaling network reveals ICAM1 phosphorylation at Tyr512 as a critical node connecting growth factor signaling (through c-MET) to oncogenic kinase activation (through SRC), with significant implications for targeted therapeutic approaches.

How does the ICAM1-SRC interaction differ between normal and pathological states?

The ICAM1-SRC interaction exhibits distinct characteristics in normal versus pathological states:

In normal physiological conditions:

• ICAM1-SRC interactions are typically transient and tightly regulated

• Baseline phosphorylation of ICAM1 at Tyr512 is maintained at low levels

• The interaction primarily serves in normal immune cell adhesion and transmigration processes

• SRC activation is controlled and limited to specific cellular responses

In pathological states (particularly cancer):

• ICAM1 expression and Tyr512 phosphorylation are significantly upregulated

• The ICAM1-SRC interaction becomes persistent, leading to constitutive SRC activation

• This sustained interaction drives oncogenic processes including EMT and angiogenesis

• The ICAM1/SRC/STAT3 positive feedback loop amplifies the aberrant signaling

• Downstream targets of SRC oncogenic signature genes show increased expression

Gene Set Enrichment Analysis (GSEA) has confirmed a strong positive correlation between ICAM1 expression and the SRC oncogenic signature in colorectal cancer patients. The expression of SRC oncogenic signature target genes is significantly decreased in ICAM1-silenced cancer cells, as revealed by quantitative RT-PCR analysis, highlighting the pathological consequence of this interaction.

What are common technical challenges when using Phospho-ICAM1 (Tyr512) Antibody and how can they be addressed?

Researchers frequently encounter several technical challenges when working with Phospho-ICAM1 (Tyr512) Antibody:

• Weak or Absent Signal:

  • Ensure samples are prepared with phosphatase inhibitors to preserve phosphorylation

  • Optimize protein extraction protocols to maintain protein integrity

  • Increase antibody concentration within the recommended range

  • Extend primary antibody incubation time (overnight at 4°C)

  • Consider enhancing detection methods (e.g., more sensitive ECL substrates)

• High Background or Non-specific Binding:

  • Increase blocking time or concentration of blocking agent

  • Optimize antibody dilution (test broader ranges)

  • Include additional washing steps with higher stringency

  • Pre-adsorb the antibody with non-specific proteins

  • Use more specific secondary antibodies

• Inconsistent Results Between Experiments:

  • Standardize lysate preparation methods

  • Control cell culture conditions that might affect phosphorylation

  • Create internal standards for normalization across experiments

  • Prepare larger batches of working solutions to reduce variability

  • Establish consistent positive controls for each experiment

• Cross-reactivity Concerns:

  • Validate specificity using blocking peptides

  • Include phospho-null mutant controls

  • Perform parallel experiments with total ICAM1 antibodies

  • Consider using multiple antibodies targeting different epitopes

Each of these challenges requires systematic troubleshooting approaches, careful optimization of experimental parameters, and rigorous controls to ensure reliable and reproducible results.

How can the Phospho-ICAM1 (Tyr512) Antibody be used in combination with other techniques to comprehensively study ICAM1 signaling?

A comprehensive investigation of ICAM1 signaling can be achieved by strategically combining the Phospho-ICAM1 (Tyr512) Antibody with complementary techniques:

• Multi-parametric Flow Cytometry:

  • Combine with total ICAM1 antibodies to simultaneously assess expression and phosphorylation status

  • Include markers for relevant signaling pathways (p-SRC, p-STAT3) to correlate phosphorylation with downstream effects

  • Analyze heterogeneity in cell populations regarding ICAM1 phosphorylation

• Phosphoproteomic Analysis:

  • Use antibody for immunoprecipitation followed by mass spectrometry

  • Identify additional phosphorylation sites and interacting proteins

  • Map the complete phosphorylation profile of ICAM1 under different conditions

• Live Cell Imaging:

  • Combine with FRET-based reporters to visualize ICAM1-SRC interactions in real-time

  • Track spatial and temporal dynamics of phosphorylation events

  • Correlate phosphorylation with changes in cell morphology and behavior

• CRISPR-Cas9 Gene Editing:

  • Generate phospho-mutant cell lines (Y512F, Y512D, etc.)

  • Confirm antibody specificity using these genetically modified cells

  • Study the functional consequences of phosphorylation in isogenic backgrounds

• Kinase Activity Assays:

  • Combine with SRC kinase assays to directly link ICAM1 phosphorylation with SRC activity

  • Use the antibody in sequential immunoprecipitation experiments to isolate phospho-ICAM1-SRC complexes

  • Measure the impact of therapeutic agents on this signaling axis

This integrated approach provides multi-dimensional insights into ICAM1 phosphorylation dynamics and its role in cellular signaling networks, enabling a more comprehensive understanding than any single technique alone.