IGSF9 Antibody

What is IGSF9 and why is it significant in research?

IGSF9 (Immunoglobulin Superfamily Member 9) is a cell membrane protein belonging to the Turtle protein family with a canonical length of 1179 amino acid residues and a molecular weight of approximately 126.6 kDa in humans. Its significance stems from its roles in dendrite outgrowth and synapse maturation, as well as its recently discovered function as an immune checkpoint regulator in cancer biology. Research indicates IGSF9 has up to two different isoforms and undergoes post-translational modifications, particularly glycosylation. The protein is also known by several synonyms including immunoglobulin superfamily member 9A, turtle homolog A, and protein turtle homolog A. Its conservation across species (with orthologs in mouse, rat, bovine, frog, chimpanzee, and chicken) suggests important evolutionary preserved functions that make it a valuable target for comparative studies .

What are the primary applications of IGSF9 antibodies in research?

IGSF9 antibodies are employed across multiple experimental techniques, with the most common applications being Flow Cytometry, ELISA, Western Blotting, and various immunohistochemistry methods (IHC, IHC-p). These antibodies are primarily utilized to detect and quantify IGSF9 expression in different cell types and tissues, particularly in cancer research where IGSF9 expression has been observed in both tumor cells and tumor-infiltrating immune cells across multiple cancer types. Beyond detection, these antibodies have therapeutic research applications, as anti-IGSF9 antibody treatment has been shown to inhibit tumor growth and enhance the efficacy of anti-PD-1 immunotherapy in experimental models .

How does IGSF9 function at the cellular and molecular level?

At the cellular level, IGSF9 serves dual functions depending on the cell type. In neural tissues, it participates in dendrite outgrowth and synapse maturation, contributing to neuronal development. In immune contexts, recent research has revealed IGSF9 functions as an immune checkpoint regulator. Mechanistically, the extracellular domain (ECD) of IGSF9 binds to T cells and inhibits their proliferation and activation, thereby potentially promoting tumor immune evasion. This function positions IGSF9 as a candidate immune checkpoint target alongside established checkpoints like PD-1/PD-L1. The mechanism appears to involve direct interaction with T cells, as experiments show that IGSF9 deficiency reduces the ability of tumor cells to suppress T-cell proliferation and results in increased tumor-infiltrating T cells in immunocompetent models .

What are the optimal sample preparation methods for IGSF9 antibody applications?

For Western Blotting applications with IGSF9 antibodies, cell lysates should be prepared using RIPA or NP-40 based lysis buffers containing protease inhibitors to prevent degradation of the relatively large (126.6 kDa) IGSF9 protein. For immunohistochemistry applications (IHC-p), optimal results are typically achieved with formalin-fixed, paraffin-embedded tissues using heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) or EDTA buffer (pH 9.0). For flow cytometry, suspension cells should be fixed with 2-4% paraformaldehyde and permeabilized with 0.1-0.5% saponin or Triton X-100 if intracellular domains are targeted. For experiments detecting IGSF9 on the cell surface, permeabilization should be avoided. Antibody dilutions should be optimized for each application, with manufacturers typically recommending starting dilutions for Western Blot (1:500-1:1000), IHC (1:100-1:200), and flow cytometry (1:50-1:100) .

How can researchers validate the specificity of IGSF9 antibodies?

Validating IGSF9 antibody specificity requires a multi-approach strategy. First, researchers should employ positive and negative controls: positive controls can utilize HEK293 cells transfected with mouse IGSF9 (as demonstrated in product validation data), while negative controls should include untransfected cells or cells transfected with irrelevant proteins. Second, antibody specificity can be confirmed through knockout/knockdown experiments, comparing IGSF9 expression in wild-type versus IGSF9-deficient samples. Third, for polyclonal antibodies targeting specific epitopes (such as those derived from synthetic peptides corresponding to AA 132-298 or AA 74-123 of human IGSF9), blocking experiments with the immunizing peptide can verify specificity. Additionally, cross-reactivity with related proteins should be assessed, especially given IGSF9's membership in the immunoglobulin superfamily. Finally, comparing results across different antibody clones targeting distinct epitopes can provide further validation .

What are the critical considerations for using IGSF9 antibodies in flow cytometry?

When using IGSF9 antibodies for flow cytometry, researchers should consider several critical factors. First, proper sample preparation is essential - for membrane-associated IGSF9 detection, gentle fixation protocols that preserve epitope accessibility are recommended. Second, titration of the antibody is crucial to determine optimal concentration that maximizes signal-to-noise ratio while minimizing background. Third, appropriate controls must be included: isotype controls (such as Rat IgG2B for clone 993107) to establish gating parameters, and fluorescence-minus-one (FMO) controls to account for spectral overlap when using multiple fluorophores. Fourth, when detecting transfected cells (as in the case of HEK293 cells transfected with mouse IGSF9), co-transfection with a reporter protein like eGFP can help identify positively transfected populations. Finally, secondary antibody selection is important - for unconjugated primary antibodies like MAB9140, compatible secondary antibodies such as APC-conjugated anti-Rat IgG (F0113) should be carefully selected to minimize cross-reactivity with other components of the experimental system .

How does IGSF9 contribute to tumor immune evasion mechanisms?

IGSF9 contributes to tumor immune evasion through multiple mechanisms based on recent research findings. Primary evidence shows that IGSF9-deficient tumor cells lose their ability to suppress T-cell proliferation and demonstrate reduced growth specifically in immunocompetent mice but not in immunocompromised models, indicating an immune-dependent mechanism. Mechanistically, the extracellular domain (ECD) of IGSF9 has been shown to directly bind to T cells and inhibit their proliferation and activation, functioning as an immune checkpoint molecule. This tumor-promoting effect of IGSF9 ECD can be reversed through CD3+ T-cell depletion, confirming the T cell-dependent nature of this mechanism. Single-cell RNA sequencing analysis reveals that IGSF9 promotes a tumor microenvironment that is favorable for tumor growth, and targeting IGSF9 shifts this environment from tumor-promoting to tumor-suppressive. Importantly, unlike other mechanisms that directly affect tumor cell proliferation, IGSF9 overexpression or knockout does not alter tumor cell proliferation in vitro or tumor growth in immunocompromised mice, further confirming its primary role in immune evasion rather than direct growth promotion .

What are the experimental approaches for studying IGSF9 in combination with other immune checkpoint inhibitors?

Studying IGSF9 in combination with other immune checkpoint inhibitors requires a systematic experimental approach. First, researchers should establish syngeneic tumor models in immunocompetent mice where both IGSF9 and established checkpoints like PD-1/PD-L1 are expressed. Sequential treatment protocols should be employed comparing: (1) control antibodies, (2) anti-IGSF9 antibodies alone, (3) established checkpoint inhibitors (e.g., anti-PD-1) alone, and (4) combination therapy with both anti-IGSF9 and established checkpoint inhibitors. Tumor growth should be monitored alongside comprehensive immune profiling using flow cytometry and single-cell RNA sequencing to assess changes in immune cell populations, activation states, and exhaustion markers. Mechanistic studies should include ex vivo T-cell functional assays (proliferation, cytokine production, cytotoxicity) from treated animals. Potential synergistic or additive effects can be quantified using tumor growth inhibition metrics and survival analysis. Additionally, potential compensatory upregulation of alternative immune checkpoints following single-agent treatment should be assessed to anticipate resistance mechanisms. This approach has already demonstrated that anti-IGSF9 antibody treatment enhances the antitumor efficacy of anti-PD-1 immunotherapy, suggesting promising potential for combination strategies .

What methodologies are effective for studying IGSF9's dual roles in neuronal development and immune regulation?

Studying IGSF9's dual roles requires integrating neuroscience and immunology techniques. For neuronal functions, primary neuronal cultures from IGSF9 wild-type and knockout mice can be analyzed for dendrite outgrowth and synapse formation using immunofluorescence microscopy with markers for dendrites (MAP2), axons (Tau), and synapses (Synaptophysin, PSD95). Time-lapse imaging can capture dynamic aspects of neuronal development. Electrophysiological recordings (patch-clamp) can assess functional synaptic maturation. For immune functions, flow cytometry can quantify IGSF9 expression on immune cell subsets, while co-culture systems of T cells with IGSF9-expressing or IGSF9-knockout cells can measure T-cell activation parameters (proliferation, cytokine production, activation markers). Recombinant IGSF9 extracellular domain can be used in binding assays to identify potential receptor(s) on T cells. To connect these functions, brain-immune interactions can be studied in IGSF9 conditional knockout mice with tissue-specific deletion. Advanced single-cell multi-omics approaches can reveal cell type-specific expression patterns and potential overlapping signaling pathways. Finally, structural biology approaches can provide insights into how IGSF9's protein domains mediate its different functions in neural and immune contexts .

How can researchers optimize Western blotting protocols for detecting IGSF9?

Optimizing Western blotting for IGSF9 detection requires addressing several challenges associated with this large membrane protein (126.6 kDa). First, sample preparation should include efficient membrane protein extraction using specialized buffers containing 0.5-1% NP-40 or Triton X-100, with extended lysis times (30-60 minutes) at 4°C. Due to IGSF9's high molecular weight, use lower percentage gels (6-8% acrylamide) and extend transfer times (overnight at low voltage or 2-3 hours at higher voltage) with the addition of 0.1% SDS in the transfer buffer to facilitate migration of large proteins. When probing, use higher antibody concentrations initially (1:250-1:500) and optimize based on results. Extended primary antibody incubation at 4°C overnight often yields better results than shorter incubations. For detection, high-sensitivity ECL substrates are recommended, especially when detecting IGSF9 in tissues with naturally lower expression levels. Importantly, as a heavily glycosylated protein, IGSF9 may show bands at higher apparent molecular weights than predicted; treatment with glycosidases can confirm glycosylation status. Finally, positive controls using lysates from cells known to express high levels of IGSF9 or recombinant IGSF9 protein should be included to validate detection methods .

What are the most effective approaches for detecting IGSF9 in different tissue types and cancer models?

Detecting IGSF9 across different tissue types and cancer models requires tailored approaches based on expression levels and tissue characteristics. For immunohistochemistry, optimal fixation protocols vary by tissue type - neural tissues benefit from shorter fixation times (24-48 hours) in 4% PFA, while more dense tissues may require longer fixation periods. Antigen retrieval methods should be optimized for each tissue type, with citrate buffer (pH 6.0) generally effective for most tissues, though some may require EDTA-based buffers (pH 9.0). For tissues with high background, blocking with 5-10% normal serum from the species of the secondary antibody is recommended, along with avidin/biotin blocking if using biotin-based detection systems. When comparing IGSF9 expression across cancer models, standardizing antibody dilutions, incubation times, and detection methods is crucial for quantitative comparisons. In heterogeneous cancers, dual immunofluorescence using IGSF9 antibodies alongside cell type-specific markers helps identify which cells express IGSF9 within the tumor microenvironment. For low-expressing samples, signal amplification methods such as tyramide signal amplification or polymer-based detection systems can enhance sensitivity. Finally, digital pathology tools for quantitative analysis provide more objective measures of expression levels across different models .

How should researchers design experiments to study IGSF9's role in T-cell suppression mechanisms?

Designing experiments to study IGSF9's role in T-cell suppression requires a systematic approach. First, in vitro co-culture assays using IGSF9-expressing cells (either natural or transfected) with isolated T cells (CFSE-labeled for proliferation tracking) can establish the direct suppressive effect. Recombinant IGSF9 extracellular domain proteins should be tested at different concentrations to establish dose-dependent relationships in T-cell suppression assays. Mechanistic studies should employ antibody blocking experiments targeting different epitopes of IGSF9 to identify regions critical for T-cell suppression. Flow cytometry panels should include markers of T-cell activation (CD25, CD69), exhaustion (PD-1, TIM-3, LAG-3), and effector function (intracellular cytokines like IFN-γ, TNF-α) to comprehensively assess IGSF9's effects. For in vivo validation, IGSF9 knockout or antibody-treated tumor models should be analyzed for changes in tumor-infiltrating lymphocyte (TIL) populations, with adoptive transfer experiments of labeled T cells to track recruitment, proliferation, and activation status in situ. Advanced techniques like imaging mass cytometry or spatial transcriptomics can provide insights into IGSF9's effects on T-cell distribution and function within the tumor microenvironment. Researchers should also investigate potential binding partners of IGSF9 on T cells through techniques like co-immunoprecipitation, proximity ligation assays, or BioID approaches .

What are the potential therapeutic applications of anti-IGSF9 antibodies in cancer immunotherapy?

Anti-IGSF9 antibodies show considerable promise as cancer immunotherapeutic agents based on several lines of evidence. Recent research demonstrates that anti-IGSF9 antibody treatment inhibits tumor growth as a monotherapy and, importantly, enhances the efficacy of established anti-PD-1 immunotherapy in preclinical models. The mechanism involves countering IGSF9's normal function as an immune checkpoint that suppresses T-cell activation and proliferation. Single-cell RNA sequencing analysis reveals that anti-IGSF9 treatment remodels the tumor microenvironment from tumor-promoting to tumor-suppressive, suggesting broader effects beyond simply blocking T-cell suppression. For clinical translation, humanized antibodies targeting specific epitopes within the IGSF9 extracellular domain would need to be developed and optimized for binding affinity, specificity, and Fc-mediated functions. Potential therapeutic applications could include both monotherapy approaches for tumors with high IGSF9 expression and combination strategies with established checkpoint inhibitors for synergistic effects. Patient stratification based on IGSF9 expression levels in tumors or immune cells might be necessary to identify those most likely to benefit from anti-IGSF9 therapy. Additionally, bispecific antibodies targeting both IGSF9 and other immune checkpoints could provide novel combination approaches in a single molecule .

How might IGSF9's role in neural development intersect with its immune functions in neurological disorders?

The dual functionality of IGSF9 in both neural development and immune regulation suggests potential significant intersections in neurological disorders. In neurodevelopmental contexts, IGSF9's established role in dendrite outgrowth and synapse maturation indicates that dysregulation could contribute to disorders characterized by aberrant neural connectivity. Simultaneously, its newly discovered immune checkpoint functions could modulate neuroimmune interactions in conditions with inflammatory components. In neurodegenerative diseases like Alzheimer's, multiple sclerosis, or Parkinson's disease, where both neural dysfunction and immune infiltration occur, IGSF9 could represent a bridge between these processes. Research approaches to investigate these intersections should include conditional knockout models with tissue-specific and temporally controlled IGSF9 deletion to distinguish developmental from acute effects. Single-cell transcriptomics of brain immune populations (microglia, infiltrating T cells) in neurological disease models with and without IGSF9 manipulation could reveal disease-specific regulation patterns. Furthermore, as neural-immune interactions are increasingly recognized in psychiatric disorders like depression and schizophrenia, IGSF9's dual functionality makes it a candidate molecule for investigation in these conditions as well. Methodologically, brain slice cultures combining neurons and immune cells with IGSF9 manipulations could provide controlled systems to study these interactions .

What novel antibody engineering approaches might enhance the utility of anti-IGSF9 antibodies in research and therapy?

Novel antibody engineering approaches could significantly expand anti-IGSF9 antibodies' research and therapeutic utility. First, domain-specific antibodies targeting particular regions of IGSF9's extracellular domain could help dissect which portions are responsible for T-cell suppression versus neuronal functions. Bispecific antibodies linking IGSF9 targeting with T-cell engaging domains (CD3) could convert an immunosuppressive signal into an activating one for cancer immunotherapy. For research applications, antibody fragments (Fab, scFv) with enhanced tissue penetration would improve immunohistochemistry in dense tissues and potentially cross blood-brain barrier for neurological applications. Site-specifically conjugated antibody-drug conjugates could deliver cytotoxic payloads to IGSF9-expressing tumor cells while sparing normal tissues. Recombinant antibodies with engineered Fc regions could modulate effector functions (ADCC, CDC, ADCP) based on therapeutic goals. For in vivo imaging, radiolabeled or fluorescently tagged anti-IGSF9 antibodies could track expression in animal models or potentially in clinical imaging. Intrabodies (intracellular antibodies) could be developed to study IGSF9's intracellular signaling pathways. Regarding therapeutic potential, pH-sensitive antibodies engineered to release from IGSF9 in endosomal compartments could enhance antibody recycling and extend half-life. Finally, antibody cocktails targeting multiple epitopes simultaneously could provide more complete blocking of IGSF9 function and potentially limit escape mechanisms in therapeutic applications .