SEMA5A Antibody

What is SEMA5A and what are its primary biological functions?

SEMA5A (human) or Sema5A (mouse) is a transmembrane protein belonging to the semaphorin family, originally identified as an axon guidance molecule. Beyond its neuronal functions, SEMA5A plays critical roles in vascular development and angiogenesis. Research demonstrates that SEMA5A can increase endothelial cell proliferation and migration while decreasing apoptosis, supporting its pro-angiogenic function . It also influences immune responses, particularly T-cell differentiation in conditions like systemic lupus erythematosus (SLE) . Additionally, SEMA5A has been implicated in cancer progression, with increased expression observed during pancreatic cancer advancement and at metastatic sites .

What are the known receptors for SEMA5A and how do they mediate signaling?

SEMA5A primarily signals through PlexinA1 and PlexinB3 receptors. Research has shown that PlexinA1 is upregulated and predominantly expressed in CD4+ T cells of SLE patients . The SEMA5A-PlexinA1 axis promotes Th17 cell differentiation via the PI3K/Akt/mTOR signaling pathway, as demonstrated in SLE studies . Additionally, SEMA5A can decrease apoptosis through Akt activation, enhance migration through Met tyrosine kinase activation, and promote extracellular matrix degradation through matrix metalloproteinase 9 (MMP9) . These diverse signaling mechanisms contribute to SEMA5A's varied biological functions.

How does SEMA5A antibody detection correlate with disease progression?

SEMA5A antibody detection has demonstrated significant correlations with disease progression in several pathological conditions. In pancreatic cancer studies, immunohistochemistry using SEMA5A antibodies has revealed increased SEMA5A expression during cancer progression, particularly at metastatic sites . In systemic lupus erythematosus, elevated serum SEMA5A levels correlate with disease activity and are implicated in kidney and blood system damage . Specific staining for SEMA5A has also been localized to plasma membranes in endocrine cells of pancreatic cancer tissue . These correlations make SEMA5A antibody detection valuable for disease monitoring and potential therapeutic targeting.

How can SEMA5A antibodies be utilized to study T-cell differentiation pathways in autoimmune disorders?

SEMA5A antibodies serve as crucial tools for investigating T-cell differentiation in autoimmune conditions. For comprehensive pathway analysis:

• First, isolate CD4+ T cells from patient samples using magnetic separation techniques with appropriate buffer systems (e.g., MACS® Separation Buffer) .

• Culture the isolated cells in pre-coated plates (anti-human CD3 antibody at 1 μg/mL) with soluble anti-human CD28 antibody (1 μg/mL) .

• Establish experimental groups with recombinant human SEMA5A (1 μg/mL) and control groups with sterile PBS .

• Assess Th17 differentiation through flow cytometry analysis of CD4+IL-17A+ cells and measure transcription factor expression (particularly RORγt) via qRT-PCR .

• Conduct knockdown experiments using PlexinA1-specific siRNA (sequence: 5'-GCAGUACUGACAACGUCAATT-3') to confirm receptor dependency .

• Evaluate downstream signaling through PI3K/Akt/mTOR pathway analysis using appropriate inhibitors and Western blotting techniques .

This methodological approach has revealed that SEMA5A specifically enhances Th17 polarization without affecting Th1 or Th2 skewing, providing insights into autoimmune disease mechanisms .

What techniques can be employed to assess SEMA5A's role in cancer cell migration and invasion?

Researchers investigating SEMA5A's influence on cancer cell migration and invasion should employ multiple complementary techniques:

• Wound Healing Assay: Culture cancer cells (e.g., AsPC-1 or T3M-4 pancreatic cancer lines) to confluence, create a standardized wound, and treat with recombinant SEMA5A at varying concentrations (50-100 ng/mL). Quantify wound closure percentage at regular intervals .

• Chemotaxis Assay: Use Boyden chambers or similar migration systems with SEMA5A (50-100 ng/mL) as a chemoattractant. Count migrated cells after appropriate incubation periods .

• Cellular Protrusion Analysis: Treat cancer cells with SEMA5A (50 ng/mL for 30 minutes) and quantify changes in cellular spreading using morphometric analysis .

• Matrix Metalloproteinase Activity: Assess MMP9 expression and activity following SEMA5A treatment using zymography and quantitative PCR .

• Signaling Pathway Analysis: Evaluate Met tyrosine kinase activation through phosphorylation status analysis .

These methods have demonstrated that SEMA5A significantly enhances pancreatic cancer cell migration (p=0.0234 for T3M-4 cells treated with 100 ng/mL SEMA5A) and promotes cellular protrusions (p=0.04), while not affecting cellular proliferation .

What is the relationship between SEMA5A expression and angiogenesis in pathological conditions?

SEMA5A plays a multifaceted role in promoting angiogenesis through several mechanisms:

• Endothelial Cell Proliferation: Recombinant extracellular domain of mouse Sema5A significantly increases endothelial cell proliferation .

• Anti-Apoptotic Effects: SEMA5A treatment decreases endothelial cell apoptosis, as demonstrated by reduced CaspACE FITC-VAD-FMK staining in HMEC-1 cells treated with 10 ng/ml SEMA5A for 24 hours .

• Gene Expression Modulation: SEMA5A treatment leads to increased expression of anti-apoptotic genes relative to pro-apoptotic genes in endothelial cells .

• Signaling Pathway Activation: SEMA5A decreases apoptosis through Akt activation while promoting migration through Met tyrosine kinase signaling .

• Extracellular Matrix Remodeling: SEMA5A induces extracellular matrix degradation through MMP9 activation, facilitating vessel formation .

These combined effects explain why Sema5A-deficient mice display defective branching of cranial vasculature, confirming its essential role in blood vessel formation during development and pathological conditions .

What are the optimal conditions for immunohistochemical detection of SEMA5A in tissue samples?

For optimal immunohistochemical detection of SEMA5A in tissue samples:

• Fixation and Processing: Use immersion-fixed, paraffin-embedded tissue sections for consistent results .

• Antibody Selection: Choose a validated monoclonal antibody such as Mouse Anti-Human Semaphorin 5A Monoclonal Antibody (e.g., Clone #914419, recognizing Glu23-Thr765) .

• Antibody Concentration: Apply at 15 μg/mL for optimal staining intensity .

• Incubation Conditions: Incubate overnight at 4°C to ensure complete antibody binding .

• Detection System: Employ an HRP-DAB detection system (such as Anti-Mouse HRP-DAB Cell & Tissue Staining Kit) for visualization .

• Counterstaining: Use hematoxylin as a nuclear counterstain to provide contrast .

• Controls: Include positive controls (pancreatic cancer tissue shows specific staining localized to plasma membrane in endocrine cells) and negative controls (omit primary antibody) .

This protocol has successfully demonstrated SEMA5A expression in pancreatic cancer tissues, with specific localization to plasma membranes of endocrine cells .

How should SEMA5A antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of SEMA5A antibodies is crucial for maintaining antibody activity and experimental reproducibility:

• Long-term Storage: Store unopened antibodies at -20 to -70°C for up to 12 months from the date of receipt .

• Post-Reconstitution Storage:

  • Short-term (up to 1 month): Store at 2 to 8°C under sterile conditions

  • Long-term (up to 6 months): Store at -20 to -70°C under sterile conditions

• Freeze-Thaw Cycles: Use a manual defrost freezer and avoid repeated freeze-thaw cycles which can denature antibodies and reduce activity .

• Reconstitution: Follow manufacturer's guidelines for reconstitution buffer and concentration .

• Working Dilutions: Determine optimal dilutions for each application through titration experiments, as these may vary depending on the specific application and tissue type .

• Sterility: Maintain sterile conditions during handling to prevent microbial contamination .

Adherence to these storage and handling guidelines ensures maximum antibody activity and consistent experimental results when working with SEMA5A antibodies.

What controls should be included when using SEMA5A antibodies in flow cytometry analysis of immune cells?

When analyzing SEMA5A or its receptors in immune cells using flow cytometry, researchers should implement a comprehensive control strategy:

• Isotype Controls: Include appropriate isotype-matched control antibodies to establish background fluorescence and non-specific binding levels .

• Fluorescence Minus One (FMO) Controls: Particularly important when analyzing Th cell subsets (Th1, Th2, Th17) to accurately set gates for CD4+IL-17A+, CD4+IFN-γ+, and CD4+IL-4+ populations .

• Unstained Controls: Essential for determining autofluorescence levels in different cell populations .

• Compensation Controls: Particularly important in multicolor flow cytometry to correct for spectral overlap between fluorophores .

• Biological Controls:

  • Positive controls: Include samples known to express SEMA5A or its receptors (e.g., PlexinA1-expressing CD4+ T cells from SLE patients)

  • Negative controls: Include cell populations not expected to express the target (e.g., CD4- cells)

• siRNA Knockdown Controls: When examining receptor dependencies, include samples where receptor expression (e.g., PlexinA1) has been knocked down using siRNA to confirm antibody specificity .

This comprehensive control strategy ensures reliable identification of SEMA5A-related signaling in immune cell populations, particularly when analyzing Th17 cell differentiation in autoimmune conditions .

How can researchers address contradictory findings in SEMA5A expression across different disease models?

When confronting contradictory findings regarding SEMA5A expression:

• Consider Tissue-Specific Expression Patterns: SEMA5A may exhibit different expression patterns depending on tissue context. For example, SEMA5A shows specific localization to plasma membranes in endocrine cells of pancreatic cancer but may display different patterns in other tissues .

• Account for Disease Heterogeneity: Within a disease like SLE, subgroups such as lupus nephritis patients may show distinct correlations between SEMA5A and inflammatory cytokines. While serum SEMA5A positively correlates with IL-17A levels in lupus nephritis patients (r=0.5604, p=0.0499), these correlations may differ in other SLE subgroups .

• Validate Using Multiple Detection Methods: Combine techniques such as ELISA, qRT-PCR, Western blotting, and immunohistochemistry to confirm expression patterns .

• Examine Receptor Expression: Discrepancies may result from variable receptor expression (PlexinA1 vs. PlexinB3) across different cell types or disease states .

• Consider Post-Translational Modifications: Investigate whether proteolytic processing by enzymes like ADAM17 affects SEMA5A detection in different contexts .

• Standardize Experimental Conditions: Use consistent cell densities, antibody concentrations, and incubation times across experiments to eliminate technical variables .

By systematically addressing these factors, researchers can reconcile contradictory findings and develop a more nuanced understanding of SEMA5A's role across different pathological conditions.

What are the key considerations when interpreting SEMA5A-mediated effects on cell migration versus proliferation?

When distinguishing between SEMA5A's effects on cell migration versus proliferation:

• Use Complementary Assays: Employ multiple assay types to differentiate between effects:

  • Migration: Wound healing assays, chemotaxis assays, and cellular protrusion analysis

  • Proliferation: MTT assays, BrdU incorporation, or Ki-67 staining

• Consider Time-Dependent Effects: SEMA5A may exhibit different effects depending on exposure duration. Short-term exposure (30 minutes) may primarily affect migration through cellular protrusions (p=0.04), while longer-term effects may impact other cellular functions .

• Concentration-Dependent Responses: Different concentrations of SEMA5A may elicit distinct cellular responses. For example, 100 ng/mL SEMA5A significantly enhances T3M-4 cell migration (p<0.001) compared to lower concentrations .

• Account for Cell Type Specificity: Research has shown that SEMA5A significantly enhances migration in pancreatic cancer cells but shows no effect on their proliferation as demonstrated by MTT assays .

• Analyze Pathway-Specific Activation: Determine whether activated signaling pathways are primarily associated with migration (Met tyrosine kinases, MMP9) versus proliferation (cell cycle regulators) .

• Control for Indirect Effects: SEMA5A's pro-angiogenic effects on endothelial cells might indirectly affect tumor cell behavior in vivo but not in isolated in vitro systems .

This methodological approach has revealed that SEMA5A predominantly affects cellular migration in pancreatic cancer models rather than proliferation, suggesting its primary role in metastatic spread rather than tumor growth .

How can researchers validate the specificity of observed SEMA5A-mediated signaling pathways?

To validate the specificity of SEMA5A-mediated signaling pathways:

• Receptor Knockdown Experiments: Utilize siRNA targeting specific receptors (e.g., PlexinA1 with sequence 5'-GCAGUACUGACAACGUCAATT-3') to confirm receptor dependency. This approach has demonstrated that PlexinA1 knockdown regulates IL-17A production by CD4+ T cells .

• Pathway Inhibitor Studies: Apply specific inhibitors of the PI3K/Akt/mTOR pathway to confirm SEMA5A's mechanism in promoting Th17 differentiation .

• Recombinant Protein Controls: Compare effects of full-length SEMA5A versus the extracellular domain to determine which protein regions mediate specific signaling outcomes .

• Dose-Response Relationships: Establish clear dose-response curves using varying concentrations of SEMA5A (50-100 ng/mL) to confirm biological relevance and specificity .

• Cross-Validation in Multiple Cell Types: Confirm pathway activation across different cellular models (e.g., AsPC-1 and T3M-4 pancreatic cancer cells; HMEC-1 endothelial cells) to establish consistency .

• Genetic Approaches: Utilize data from Sema5A-deficient mice, which display defective branching of cranial vasculature, to validate in vitro findings in physiological contexts .

• Temporal Analysis: Examine signaling events at multiple time points to distinguish between primary and secondary pathway activation .

This comprehensive validation approach ensures that observed SEMA5A-mediated effects are specific to the protein and provides robust evidence for its signaling mechanisms across different biological contexts.