ITGA9 Antibody

What is ITGA9 and why is it significant in cancer research?

ITGA9 (Integrin alpha9) is one of the less studied integrin subunits that facilitates accelerated cell migration and regulates diverse biological functions including angiogenesis, lymphangiogenesis, cancer cell proliferation and migration. Its significance in cancer research has grown substantially as studies have demonstrated its essential role in metastatic dissemination in multiple cancer types including rhabdomyosarcoma, neuroblastoma, and breast cancer . ITGA9 represents an important research target because metastatic progression accounts for more than 90% of cancer deaths, yet there remains a lack of viable pharmacological options to reduce metastasis formation . The protein typically functions by forming heterodimers with integrin beta1 (ITGB1), although recent research suggests potential interactions with other beta subunits including ITGB7 .

How do I select the appropriate anti-ITGA9 antibody for my research?

When selecting an anti-ITGA9 antibody, researchers should consider several critical factors. First, determine whether you need a polyclonal or monoclonal antibody based on your specific application requirements. For detection of total ITGA9 protein, antibodies targeting conserved epitopes are preferable. The choice of antibody should also be informed by the specific application (western blot, immunohistochemistry, flow cytometry, functional studies). Research has demonstrated that primer and antibody choice is particularly important for ITGA9 investigation, as 11% of breast cancer samples showed different ITGA9 expression depending on which primer pairs were used . This suggests potential involvement of genetic changes or alternative splicing that could affect antibody recognition sites. For function-blocking studies, antibodies such as Y9A2 have been used successfully in human cell lines, though this particular antibody does not cross-react with mouse ITGA9 . Always validate the antibody in your experimental system before proceeding with full studies.

What are the best positive and negative controls for ITGA9 antibody experiments?

Establishing appropriate controls is essential for ITGA9 antibody experiments. For positive controls, cell lines with documented ITGA9 expression include HT1080 cells and Tera-2 cells, which express ITGA9-ITGB1 . RPMI 8866 cells have been shown to express ITGA9 that may pair with ITGB7 . Tissues known to express ITGA9 can also serve as positive controls. For negative controls, consider using cells where ITGA9 has been knocked down via shRNA methods as demonstrated in research on rhabdomyosarcoma and neuroblastoma cell lines . When performing immunohistochemistry, include an isotype control antibody at the same concentration as your anti-ITGA9 antibody. For functional studies, competitive inhibition with synthetic peptides containing the MLD sequence (which blocks ITGA4/ITGA9 interactions) can provide validation of antibody specificity . Additional negative controls include cells known to lack ITGA9 expression or tissues where ITGA9 expression has been comprehensively characterized as absent.

How should I optimize western blot protocols for ITGA9 detection?

Optimizing western blot protocols for ITGA9 detection requires careful consideration of several parameters. ITGA9 is a relatively large protein (approximately 140-150 kDa), so use lower percentage gels (7-8% polyacrylamide) to achieve proper separation. For protein extraction, use lysis buffers containing protease inhibitors to prevent degradation of the integrin proteins. Research protocols have demonstrated effective ITGA9 detection using standard western blot techniques with appropriate antibody dilutions . When analyzing phosphorylation-dependent signaling downstream of ITGA9, such as FAK phosphorylation at Tyr397, include phosphatase inhibitors in your lysis buffer . For loading controls, total FAK levels may remain unchanged while phosphorylation states vary significantly in response to ITGA9 manipulation, as demonstrated in studies of rhabdomyosarcoma and neuroblastoma cells . Verification of antibody specificity can be achieved through rescue experiments where ITGA9 expression is restored in knockdown cells, resulting in the reappearance of the specific band on western blots .

What RT-PCR methodologies are recommended for analyzing ITGA9 expression?

For analyzing ITGA9 expression via RT-PCR, consider both conventional and quantitative approaches. For conventional RT-PCR, multiplex methods using GAPDH or β-actin as internal controls have been successfully employed . Use DNase treatment of RNA samples before cDNA synthesis to eliminate genomic DNA contamination. Research protocols have successfully amplified ITGA9 using the following conditions: 94°C for 4 min initial denaturation, followed by cycles of 94°C for 30 sec, 61°C (for ITGA9 1835/2935 primers) or 64°C (for ITGA9 150/960 primers) for 60 sec, and 72°C for 1 min, with a final elongation at 72°C for 10 min . For quantitative real-time PCR, TaqMan assays have been effectively used with primers targeting conserved regions of the ITGA9 transcript (e.g., 5′-GTT GGT GGG AAT CCT CAT CTT C-3′ and 5′-TTT GTA CCT TCG GCG AAA GAA-3′) . Importantly, research has shown that different primer pairs may yield varying results in some samples, suggesting potential alternative splicing or genetic alterations in ITGA9 . Therefore, using multiple primer sets targeting different regions of the transcript is advisable for comprehensive analysis.

How can I effectively use ITGA9 antibodies in cell adhesion assays?

Cell adhesion assays using ITGA9 antibodies require careful experimental design. First, coat plates with purified ADAM proteins (particularly ADAM2) or other known ITGA9 ligands at an optimal concentration (typically 5-10 μg/ml) . Block non-specific binding sites with BSA or appropriate blocking solution. For antibody-based inhibition studies, preincubate cells with function-blocking anti-ITGA9 antibodies (such as Y9A2 for human cells) at concentrations ranging from 5-20 μg/ml for 15-30 minutes before adding to the coated plates . Include appropriate controls such as isotype-matched control antibodies. To distinguish ITGA9-specific adhesion from other integrin-mediated interactions, include comparative conditions with function-blocking antibodies against other integrin subunits (e.g., anti-ITGA4, anti-ITGB1, anti-ITGB7) alone and in combination . Research has shown that combining anti-ITGA9 and anti-ITGA4 antibodies can produce additive inhibitory effects in certain cell types, suggesting cooperative or redundant functions . Adhesion-blocking peptides containing the MLD motif (recognized by ITGA4/ITGA9 integrins) can serve as additional controls to validate the specificity of ITGA9-mediated adhesion .

How can I assess ITGA9-mediated signaling pathways in cancer metastasis models?

Assessing ITGA9-mediated signaling pathways in cancer metastasis models requires a multi-faceted approach. Begin by establishing baseline ITGA9 expression in your cancer model through western blot and RT-PCR. Then manipulate ITGA9 expression through genetic approaches (shRNA knockdown or overexpression) as demonstrated in rhabdomyosarcoma and neuroblastoma studies . To analyze downstream signaling, focus on focal adhesion kinase (FAK) phosphorylation at Tyr397, which shows significant reduction following ITGA9 downregulation while total FAK levels remain unchanged . This can be assessed via western blot using phospho-specific antibodies. For in vivo metastasis models, consider both spontaneous and experimental metastasis assays, injecting cells with modulated ITGA9 expression and quantifying metastatic burden through appropriate imaging techniques. To evaluate pharmacological inhibition of ITGA9, synthetic peptides mimicking key interaction domains (such as those found in ADAM proteins) have shown promise in reducing metastasis in multiple cancer models . Complement these approaches with cell migration and invasion assays to correlate in vitro behavior with in vivo metastatic potential. For comprehensive pathway analysis, consider phospho-proteomic approaches to identify additional signaling nodes affected by ITGA9 manipulation.

What methods can detect novel ITGA9 heterodimeric partners beyond the established ITGA9-ITGB1 complex?

Detecting novel ITGA9 heterodimeric partners requires sophisticated biochemical and cell biological approaches. First, conduct co-immunoprecipitation experiments using anti-ITGA9 antibodies followed by mass spectrometry analysis to identify associated proteins. Research has suggested that ITGA9 may partner with ITGB7 in addition to the well-established ITGB1 partner, particularly in RPMI 8866 cells that lack detectable ITGB1 on the cell surface . To confirm suspected heterodimers, perform reciprocal co-immunoprecipitations with antibodies against candidate beta subunits. Proximity ligation assays (PLA) can provide visual confirmation of protein-protein interactions within intact cells. For functional validation, employ siRNA knockdown of candidate beta subunits (e.g., ITGB7) and assess the impact on ITGA9-dependent functions such as cell adhesion to known ligands . Cross-linking studies with membrane-impermeable cross-linkers before immunoprecipitation can stabilize transient interactions. Additionally, recombinant expression of tagged versions of ITGA9 and candidate beta subunits in heterologous systems can confirm the ability to form stable complexes. Flow cytometry using conformation-specific antibodies can further distinguish between free and dimerized integrin subunits on the cell surface.

How can ITGA9 antibodies be used to study the relationship between epigenetic silencing and ITGA9 expression in cancer?

ITGA9 antibodies can be valuable tools for studying the relationship between epigenetic silencing and ITGA9 expression in cancer. Begin with methyl-specific PCR (MSP) to assess the methylation status of ITGA9 CpG islands using primers specific to methylated and unmethylated DNA sequences . Correlate methylation patterns with ITGA9 protein expression levels determined by western blot or immunohistochemistry using validated anti-ITGA9 antibodies. To establish causality, treat cells with DNA methyltransferase inhibitors (such as 5-aza-2'-deoxycytidine) and histone deacetylase inhibitors (such as trichostatin A), then measure changes in ITGA9 expression at both mRNA and protein levels. For comprehensive epigenetic profiling, combine MSP with bisulfite sequencing, chromatin immunoprecipitation (ChIP) to analyze histone modifications at the ITGA9 promoter, and ATAC-seq to assess chromatin accessibility. In clinical samples, use immunohistochemistry with anti-ITGA9 antibodies to correlate protein expression with patient data and methylation analysis of the same samples. Research has indicated that epigenetic mechanisms may contribute to altered ITGA9 expression in various cancers, making this a valuable area of investigation for understanding the regulation of this important integrin .

How should I address inconsistent ITGA9 detection results across different experimental platforms?

Inconsistent ITGA9 detection across experimental platforms may stem from multiple factors that require systematic troubleshooting. First, consider potential alternative splicing or genetic alterations in your samples, as research has shown that 11% of breast cancer samples exhibited different ITGA9 expression patterns depending on which primer pairs were used . This suggests that primers or antibodies targeting different regions of ITGA9 may yield different results. Verify antibody specificity through positive and negative controls, including ITGA9 knockdown cells. For western blotting inconsistencies, optimize protein extraction conditions, as membrane proteins like integrins may require specialized lysis buffers. If discrepancies exist between mRNA and protein levels, consider post-transcriptional regulation mechanisms. For immunostaining applications, compare different fixation methods, as some epitopes may be sensitive to specific fixatives. When comparing results across cell lines or tissue samples, account for heterogeneity in ITGA9 expression and potential context-dependent regulation. If using function-blocking antibodies in adhesion assays, titrate antibody concentrations carefully, as different experimental systems may require different optimal concentrations . Finally, when interpreting results, consider the heterodimeric nature of integrins—ITGA9 function depends on its beta subunit partner, which may vary across cell types .

What could explain contradictory results between ITGA9 mRNA expression and protein detection?

Contradictory results between ITGA9 mRNA expression and protein detection can arise from several biological and technical factors. Post-transcriptional regulation mechanisms, including microRNA-mediated suppression or altered mRNA stability, may result in reduced protein expression despite abundant transcript levels. Research on RPMI 8866 cells has demonstrated detectable ITGB1 mRNA but absence of ITGB1 protein on the cell surface, suggesting that translation, trafficking, or stability issues can affect integrin expression . Alternative splicing events may produce transcript variants that are detected by some PCR primers but not others, while the corresponding protein isoforms might not be recognized by certain antibodies . Epigenetic modifications can affect both transcription and translation efficiency. For technical considerations, ensure that your protein extraction method effectively solubilizes membrane proteins like ITGA9, and verify antibody specificity using appropriate controls. Consider the sensitivity differences between RT-PCR (which can detect low abundance transcripts) and western blotting or immunostaining (which have different detection thresholds for proteins). Timing differences between mRNA induction and protein accumulation may also contribute to apparent discrepancies. To resolve these issues, employ multiple detection methods targeting different regions of both the transcript and protein, and consider pulse-chase experiments to assess protein stability.

How can I validate that functional effects observed with anti-ITGA9 antibodies are specific?

Validating the specificity of functional effects observed with anti-ITGA9 antibodies requires multiple complementary approaches. First, perform parallel experiments using genetic manipulation of ITGA9 expression (shRNA knockdown or CRISPR/Cas9 knockout) to confirm that antibody-mediated effects match those produced by reducing ITGA9 protein levels . Include appropriate isotype control antibodies at equivalent concentrations to rule out non-specific effects. For function-blocking studies, use competitive inhibition with synthetic peptides containing recognition motifs for ITGA9 (such as MLD peptides) as an alternative method of inhibition . The convergence of results from antibody treatment and peptide competition strongly supports specificity. To further confirm target engagement, perform cellular thermal shift assays (CETSA) to demonstrate direct binding of antibodies or inhibitory peptides to ITGA9, as has been done with the RA08 peptide . Rescue experiments in which ITGA9 expression is restored in knockdown cells should reverse the effects of genetic ITGA9 reduction and overcome antibody inhibition if additional ITGA9 protein is expressed above the neutralizing capacity of the antibody . Finally, dose-response studies with increasing concentrations of anti-ITGA9 antibodies should show proportional functional effects, plateauing at saturating concentrations.

How might ITGA9 antibodies be utilized in developing anti-metastatic therapies?

ITGA9 antibodies hold significant potential for developing anti-metastatic therapies through multiple strategies. Function-blocking antibodies could directly interfere with ITGA9-mediated cell adhesion and migration, critical processes in metastatic cascade. Research has demonstrated that pharmacological inhibition of ITGA9 with a synthetic peptide simulating a key interaction domain of ADAM proteins effectively reduced metastasis in experimental models of rhabdomyosarcoma, neuroblastoma, and breast cancer . Antibody-drug conjugates (ADCs) targeting ITGA9 could deliver cytotoxic payloads specifically to ITGA9-expressing cancer cells with metastatic potential. For developing such therapies, researchers should first characterize ITGA9 expression patterns across primary tumors and their metastases to identify high-expression cancer types amenable to ITGA9-targeted therapy. Humanized anti-ITGA9 antibodies with optimized binding properties and appropriate effector functions would be required for clinical translation. Combination approaches targeting both ITGA9 and its signaling partners (such as FAK) might provide synergistic anti-metastatic effects, as research has shown that ITGA9 downregulation reduces FAK phosphorylation . Careful evaluation of potential off-target effects is essential, as ITGA9 plays roles in normal physiological processes including lymphangiogenesis. Preclinical studies should assess both efficacy in preventing new metastasis formation and effects on established metastatic lesions.

What role might ITGA9 play in the tumor microenvironment beyond cancer cell functions?

ITGA9's role in the tumor microenvironment likely extends beyond direct effects on cancer cells, presenting rich opportunities for investigation using ITGA9 antibodies. ITGA9 is known to regulate angiogenesis and lymphangiogenesis, processes crucial for tumor progression and metastatic dissemination . Researchers can use anti-ITGA9 antibodies to study ITGA9 expression and function in endothelial cells, lymphatic vessels, and stromal components within the tumor microenvironment. Immunohistochemical analysis with anti-ITGA9 antibodies can map expression patterns across different cellular compartments in tumor samples. ITGA9 may mediate interactions between cancer cells and extracellular matrix components or ADAM family proteins expressed by stromal cells . Function-blocking antibodies could be used in co-culture systems to dissect these heterotypic cellular interactions. ITGA9 might also influence immune cell recruitment and function within tumors, as integrins are known to regulate immune cell trafficking and activation. Single-cell analyses combining anti-ITGA9 antibodies with markers for various stromal and immune cell populations could provide insights into cell type-specific roles. Additionally, spatial transcriptomics and proteomics approaches incorporating ITGA9 detection could reveal microenvironmental niches where ITGA9 signaling is particularly active, potentially identifying new therapeutic opportunities beyond direct targeting of cancer cells.

How can advanced imaging techniques be combined with ITGA9 antibodies to track metastatic processes in real-time?

Combining advanced imaging techniques with ITGA9 antibodies offers powerful approaches for tracking metastatic processes in real-time. Researchers can develop fluorescently labeled anti-ITGA9 antibodies or antibody fragments (Fabs) that retain binding specificity while minimizing interference with function. These can be used with intravital microscopy in animal models to visualize ITGA9-expressing cells during metastatic spread. For longer-term studies, consider antibodies conjugated to near-infrared fluorophores for whole-body imaging in preclinical models. Super-resolution microscopy techniques like STORM or PALM combined with fluorescently labeled anti-ITGA9 antibodies can reveal nanoscale organization of ITGA9 in membrane microdomains during cell migration and invasion. Multiplexed imaging approaches can simultaneously track ITGA9 and its binding partners or downstream signaling components to correlate integrin dynamics with functional outcomes. For clinical translation, radiolabeled anti-ITGA9 antibodies could potentially be developed for PET imaging to detect metastasis-prone tumor cells. In ex vivo settings, light-sheet microscopy of cleared tissue samples labeled with anti-ITGA9 antibodies can provide three-dimensional visualization of metastatic niches. Complementary to these approaches, biosensors based on FRET technology could be designed to detect ITGA9 activation states, offering insights into not just localization but functional status during metastatic progression. Given ITGA9's established role in metastasis in multiple cancer types, these imaging approaches could significantly advance our understanding of metastatic mechanisms .