Cleaved-ITGAV (K889) Antibody

What is Cleaved-ITGAV (K889) Antibody and what epitope does it recognize?

Cleaved-ITGAV (K889) Antibody is a rabbit polyclonal antibody that specifically recognizes the cleaved form of Integrin Alpha-V at the K889 position. The antibody binds to the endogenous Integrin Alpha-V at the amino acid region 810-890 in the C-terminal domain. It is generated against a synthesized peptide derived from human ITGAV at the amino acid range 840-889. This specificity makes it valuable for detecting proteolytic processing of ITGAV, which often occurs during cellular signaling events relevant to cancer progression .

What are the primary applications for Cleaved-ITGAV (K889) Antibody in research?

The primary validated applications for Cleaved-ITGAV (K889) Antibody include:

The antibody has been particularly validated in cancer cell lines, demonstrating specific binding to the cleaved form of ITGAV after treatments that induce proteolytic cleavage (such as etoposide treatment at 25μM for 1 hour in A549 cells) .

How should Cleaved-ITGAV (K889) Antibody be stored and handled to maintain optimal activity?

For optimal performance of Cleaved-ITGAV (K889) Antibody:

• Store the antibody at -20°C for up to 1 year from the date of receipt

• Avoid repeated freeze-thaw cycles which can compromise antibody performance

• The antibody is formulated in PBS with 50% Glycerol, 0.5% BSA, and 0.02% Sodium Azide as preservatives

• Allow the antibody to equilibrate to room temperature before opening the vial

• Centrifuge briefly before use to collect solution at the bottom of the vial

Following these storage guidelines ensures antibody stability and reproducible experimental results across multiple uses .

What controls should be included when using Cleaved-ITGAV (K889) Antibody in Western blotting?

When conducting Western blot experiments with Cleaved-ITGAV (K889) Antibody, the following controls are essential:

• Positive control: Lysates from A549 cells treated with etoposide (25μM for 1h) to induce ITGAV cleavage

• Negative control: Lysates from untreated cells showing minimal cleaved ITGAV

• Peptide blocking control: Preincubation of the antibody with the immunizing peptide to confirm specificity

• Loading control: Detection of housekeeping proteins (β-actin, GAPDH) to normalize protein loading

• Secondary antibody control: Omitting primary antibody to verify no non-specific binding from secondary antibody

Western blot analysis data demonstrates that blocking with the synthesized peptide effectively eliminates the specific band, confirming antibody specificity .

How can Cleaved-ITGAV (K889) Antibody be used to investigate the role of ITGAV in cancer progression and metastasis?

Cleaved-ITGAV (K889) Antibody serves as a powerful tool for investigating ITGAV's role in cancer progression through several advanced approaches:

• Comparative expression analysis: Detection of cleaved ITGAV levels across cancer cell lines with different invasive potentials (e.g., high-invasive UM1 and UMSCC-5 vs. low-invasive UM2 and UMSCC-6 in HNSCC)

• siRNA knockdown validation: Confirming ITGAV knockdown efficiency in functional studies investigating migration, invasion, and proliferation

• Treatment response monitoring: Assessing changes in ITGAV cleavage following drug treatments or genetic manipulations

• Patient sample analysis: Correlating cleaved ITGAV levels with clinical outcomes, tumor stage, and metastatic potential

Research utilizing similar approaches has demonstrated that ITGAV is significantly overexpressed in HNSCC, with knockdown experiments confirming its role in promoting cell migration, invasion, viability, and proliferation. Western blot analysis revealed significantly higher ITGAV expression in high-invasive cell lines compared to low-invasive cell lines, with UM1 showing a 44.5-fold increase over UM2 and a 53-fold increase over UMSCC-6 .

What is the significance of ITGAV cleavage at K889 in tumor biology and how can the antibody help elucidate this process?

The cleavage of ITGAV at K889 represents a post-translational modification that impacts integrin signaling and function in tumor biology. The Cleaved-ITGAV (K889) Antibody helps elucidate this process by:

• Specifically detecting the cleaved form, allowing researchers to distinguish between intact and processed ITGAV

• Enabling temporal studies of ITGAV cleavage during cancer progression

• Facilitating investigation of the proteases responsible for K889 cleavage

• Supporting studies on downstream signaling effects of ITGAV cleavage

This specific cleavage event may be mechanistically linked to ITGAV's role in promoting tumor progression. Research has established that ITGAV contributes significantly to cell adhesion by integrating into the extracellular matrix, with its gene product forming heterodimers with various β subunits including the αVβ3 integrin. These molecular complexes are involved in cell adhesion, migration, proliferation, and signal transduction—all processes critical to cancer progression .

What are the optimal conditions for detecting cleaved ITGAV in various cancer cell models?

Optimizing detection of cleaved ITGAV across cancer models requires careful consideration of several experimental parameters:

When comparing expression across cell lines, it's crucial to normalize to loading controls and include positive controls (e.g., A549 cells treated with etoposide). Western blot analysis of ITGAV expression in HNSCC cell lines revealed significantly higher levels in high-invasive lines compared to low-invasive lines, with quantitative differences confirmed by qPCR analysis showing 21.5-53 fold increases in expression .

How can researchers troubleshoot non-specific binding or low signal issues when using Cleaved-ITGAV (K889) Antibody?

When encountering technical challenges with Cleaved-ITGAV (K889) Antibody, consider the following systematic troubleshooting approaches:

• For non-specific binding:

  • Increase blocking time (2 hours at room temperature)

  • Use alternative blocking agents (switching between milk and BSA)

  • Include 0.1-0.3% Triton X-100 in antibody dilution

  • Perform peptide competition assay to identify specific bands

  • Increase washing duration and number of washes

• For low signal issues:

  • Reduce antibody dilution (1:500 instead of 1:1000)

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

  • Use signal enhancement systems (amplified chemiluminescence)

  • Check protein extraction efficiency with alternative lysis buffers

  • Enrich for membrane fractions where integrins predominantly localize

• For inconsistent results:

  • Standardize lysate preparation methods across experiments

  • Use freshly prepared samples when possible

  • Consider phosphatase inhibitors in addition to protease inhibitors

  • Validate antibody performance with positive control lysates

Western blot analysis from published studies demonstrates the specificity of ITGAV antibodies when using proper controls, such as peptide blocking experiments where the lane treated with the synthesized peptide shows elimination of the specific band .

What is the current evidence for ITGAV as a prognostic biomarker in cancer research?

Multiple studies have established ITGAV as a potential prognostic biomarker across various cancer types:

• Head and Neck Squamous Cell Carcinoma (HNSCC): Analysis of TCGA datasets revealed significantly upregulated ITGAV gene expression in HNSCC. Patients with higher ITGAV expression demonstrated worse survival outcomes compared to those with lower expression levels .

• Digestive System Cancers: Comprehensive analysis across multiple cancer types showed ITGAV upregulation correlated with poor prognosis in:

  • Stomach adenocarcinoma (STAD)

  • Liver hepatocellular carcinoma (LIHC)

  • Pancreatic adenocarcinoma (PAAD)

  • Esophageal carcinoma (ESCA)

  • Colorectal carcinoma (COAD)

• Other Cancer Types: Similar patterns were observed in bladder urothelial carcinoma (BLCA), lung squamous cell carcinoma (LUSC), and sarcoma (SARC), suggesting ITGAV may serve as a broad-spectrum cancer biomarker .

How can Cleaved-ITGAV (K889) Antibody contribute to understanding the mechanistic role of ITGAV in immune regulation within the tumor microenvironment?

Cleaved-ITGAV (K889) Antibody offers unique opportunities to investigate the intersection between ITGAV processing and immune regulation in the tumor microenvironment:

• Immune Cell Interaction Studies: The antibody can detect cleaved ITGAV on tumor cells interacting with immune cells, potentially revealing how proteolytic processing affects immune recognition

• Extracellular Matrix (ECM) Remodeling: Detecting cleaved forms can help elucidate how ITGAV processing contributes to ECM remodeling, which impacts immune cell infiltration and function

• Immunoregulatory Signaling: By tracking cleaved ITGAV in co-culture systems of tumor and immune cells, researchers can investigate downstream signaling pathways affecting immunosuppression

• Therapeutic Response Monitoring: The antibody can assess how immunotherapies affect ITGAV processing, potentially identifying mechanisms of resistance or response

Recent research highlights ITGAV's multifaceted role across diverse cancer types, with bioinformatic analyses revealing connections to immune infiltration levels. This suggests cleaved ITGAV may play a significant role in shaping the tumor immune microenvironment, making it a valuable target for immunotherapeutic development strategies .

What functional assays can be performed following ITGAV knockdown to validate its role in cancer progression?

Following ITGAV knockdown using siRNA techniques, several functional assays can validate its role in cancer progression:

Experimental validation of these approaches has been demonstrated in HNSCC cell lines, where siRNA knockdown of ITGAV significantly reduced migration capacity, invasion capabilities, cell viability, and colony formation ability in both UM1 and UMSCC-5 high-invasive cell lines .

How can researchers verify the specificity of Cleaved-ITGAV (K889) Antibody for the cleaved form versus the intact protein?

Verifying the specificity of Cleaved-ITGAV (K889) Antibody requires multiple complementary approaches:

• Peptide Competition Assay: Preincubate antibody with excess immunizing peptide before Western blotting. Specific bands should disappear while non-specific bands remain.

• Molecular Weight Verification: The intact ITGAV protein (approximately 150 kDa) versus the cleaved C-terminal fragment (expected lower molecular weight) should be distinguishable on Western blots.

• Induction of Cleavage: Compare samples with and without treatments known to induce ITGAV cleavage (e.g., etoposide treatment).

• siRNA Knockdown: Transfection with ITGAV siRNA should reduce both intact and cleaved forms, confirming the band's identity.

• Recombinant Protein: Using purified recombinant ITGAV protein as a positive control and size reference.

Western blot data from validated studies shows that when blocking with the synthesized peptide, the specific band disappears, providing confirmation of antibody specificity. Additionally, treatment of A549 cells with etoposide (25μM for 1h) induces cleavage that can be specifically detected, further validating the antibody's selectivity for the cleaved form .