SEMA6B Antibody

What is SEMA6B and what are its key biological functions?

SEMA6B (sema domain, transmembrane domain, and cytoplasmic domain, semaphorin 6B) is a 95 kDa protein consisting of 888 amino acids that belongs to the semaphorin family . Recent research has demonstrated that SEMA6B plays significant roles in:

• Macrophage-mediated inflammatory responses, with overexpression activating systemic inflammatory pathways

• Hepatocyte apoptosis regulation, where elevated expression can inhibit cell proliferation through G0/G1 cell cycle arrest

• Immune cell infiltration in colorectal cancer (CRC), showing strong correlations with CD4+ T cells, macrophages, neutrophils, and dendritic cells

• Serving as a potential biomarker for Hepatitis B virus-associated acute-on-chronic liver failure (HBV-ACLF), with significantly higher expression in non-survivors

Understanding these functions is essential for researchers designing experiments targeting inflammation, liver disease, or cancer immunology pathways involving SEMA6B.

What types of SEMA6B antibodies are available for research applications?

Researchers have access to several types of SEMA6B antibodies with different applications:

When selecting an antibody, researchers should consider their specific experimental needs. For example:

• Unconjugated antibodies are versatile for various detection methods

• HRP-conjugated antibodies are optimal for ELISA and immunoblotting

• FITC-conjugated antibodies are ideal for flow cytometry and fluorescence microscopy

• Biotin-conjugated antibodies offer signal amplification capability through streptavidin interactions

What are the recommended protocols for SEMA6B antibody validation?

Proper validation of SEMA6B antibodies is critical for ensuring experimental rigor. A comprehensive validation approach should include:

• Western blot analysis: Confirm specificity by observing a single band at approximately 95 kDa when testing in human samples. This was the approach used to validate SEMA6B knockdown and overexpression efficiencies in RAW264.7 and AML12 cell models .

• Positive and negative controls:

  • Positive controls: Use tissues known to express SEMA6B (e.g., liver tissues from HBV-ACLF patients)

  • Negative controls:

    • SEMA6B knockout cells created using CRISPR/Cas9 technology

    • SEMA6B knockdown cells using siRNA targeting SEMA6B

    • IgG controls from the same host species as the antibody

• Cross-reactivity testing: While the antibodies in search results show reactivity with human samples , researchers should validate cross-reactivity if working with other species.

• Functional validation: Confirm that the antibody detects functional changes in SEMA6B, such as in pull-down assays for identifying SEMA6B-interacting proteins using LC-MS/MS, as described in the proteomic analysis methods .

How can SEMA6B antibodies be effectively used in studying inflammatory pathways?

SEMA6B has been identified as a significant mediator of inflammatory responses. To effectively study these pathways using SEMA6B antibodies:

• Co-immunoprecipitation studies: Use SEMA6B antibodies to pull down protein complexes and identify interaction partners in inflammatory signaling cascades. This approach was successfully employed to identify proteins interacting with SEMA6B in RAW264.7 cells using antibody-mediated pull-down followed by LC-MS/MS analysis .

• Cytokine profiling: After SEMA6B manipulation (overexpression or knockdown):

  • Collect cell culture supernatants

  • Use antibody arrays (such as Mouse/Human Inflammation Array) to detect multiple inflammatory cytokines simultaneously

  • Look specifically for changes in TNF-α, IL-6, IL-1β, IL-1α, and IL-10 levels

• Tissue analysis protocol:

  • Use SEMA6B antibodies for immunohistochemistry (IHC) at recommended dilutions of 1:20-1:200

  • Compare expression between inflamed and normal tissues

  • Correlate with macrophage markers to investigate SEMA6B's role in macrophage-mediated inflammation

• Gene expression correlation analysis: Combine antibody-based protein detection with qRT-PCR analysis of proinflammatory cytokines to establish correlations between SEMA6B levels and inflammatory gene expression .

What methodological approaches should be used when studying SEMA6B's role in cancer progression?

Research indicates that SEMA6B overexpression correlates with poor prognosis in colorectal cancer. To investigate this association:

• Tissue microarray analysis:

  • Use SEMA6B antibodies at 1:20-1:200 dilution for IHC

  • Quantify expression levels across cancer stages

  • Compare tumor tissues with adjacent normal tissues

  • Correlate expression with patient survival data

• Immune infiltration analysis:

  • Utilize multicolor immunofluorescence with FITC-conjugated SEMA6B antibodies (1:20-1:200)

  • Co-stain for markers of:

    • CD4+ T cells

    • Macrophages

    • MDSCs (myeloid-derived suppressor cells)

    • Tregs (regulatory T cells)

    • Neutrophils

    • Dendritic cells

• ESTIMATE algorithm implementation: Use this computational method to:

  • Calculate stromal scores, immune scores, and tumor purity

  • Correlate these metrics with SEMA6B expression

  • This approach revealed positive correlations between SEMA6B expression and stromal/immune scores, while showing negative correlation with tumor purity in colorectal cancer

• Functional studies in cancer cell lines:

  • Create SEMA6B knockdown cancer cells using siRNA

  • Assess effects on proliferation, migration, and invasion

  • Measure expression of immunosuppressive molecules before and after knockdown

  • Research has shown that SEMA6B knockdown significantly inhibits these processes in colon cancer cells

How can researchers address contradictory data when studying SEMA6B in different disease contexts?

When encountering contradictory results regarding SEMA6B function across different experimental systems:

• Context-dependent analysis framework:

  • Compare expression patterns across different tissues and cell types

  • Document differences in SEMA6B function between:

    • Liver diseases (where it mediates inflammation and hepatocyte apoptosis)

    • Colorectal cancer (where it correlates with immunosuppressive microenvironment)

• Pathway intersection analysis:

  • Identify common signaling pathways affected by SEMA6B across disease models

  • Focus on inflammatory mediators that consistently change with SEMA6B manipulation

  • Document cell-type specific effects (e.g., effects in macrophages versus hepatocytes)

• Antibody validation across experimental systems:

  • Verify antibody specificity in each tissue/cell type being studied

  • Use multiple antibody clones to confirm findings

  • Include appropriate positive and negative controls specific to each experimental system

• Integrated multi-omics approach:

  • Combine antibody-based detection with transcriptomics data

  • Validate protein-level findings with mRNA expression analysis

  • Compare proteomics and transcriptomics results to identify discrepancies that might explain contradictory findings

What are the optimal conditions for using SEMA6B antibodies in various immunoassays?

For optimal results with SEMA6B antibodies across different applications:

Storage recommendations:

• Store antibodies at -20°C

• Aliquoting is unnecessary for -20°C storage

• Antibodies remain stable for one year after shipment

• Storage buffer typically contains PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

For SEMA6B protein detection in complex biological samples, researchers should optimize blocking conditions, antibody concentrations, and incubation times for their specific experimental system.

How should researchers design SEMA6B knockdown/knockout experiments to study its function?

Based on successful approaches in the literature:

• CRISPR/Cas9-mediated knockout:

  • Clone single guide RNAs (sgRNAs) targeting SEMA6B into lenti-CRISPRv2 vector

  • Produce viral stocks by co-transfecting modified plasmids with pMD2.G and psPAX2 into 293T cells

  • Infect target cells (e.g., RAW264.7) with collected virus

  • Select stable knockdown cells using puromycin for approximately 1 week

  • Verify knockout efficiency by western blot analysis

• siRNA-mediated knockdown:

  • Use human siRNA targeting SEMA6B (20 nmol/L)

  • Transfect using Lipofectamine RNAi Max

  • This approach has been successfully applied to THP1 and HepaRG cells

  • Validate knockdown efficiency using western blot analysis

• Overexpression system design:

  • Transfect cells with lentiviruses encoding SEMA6B (commercially produced)

  • Establish stable overexpressing cell lines

  • Validate overexpression using western blot with SEMA6B antibodies

• In vivo knockout model:

  • Generate SEMA6B knockout mice

  • Induce disease models (e.g., liver failure using LPS/D-gal)

  • Compare phenotypes between wild-type and knockout animals

  • Assess key parameters:

    • Inflammatory cytokine levels (TNF-α, IL-6, IL-1β, IL-1α, IL-10)

    • Apoptosis markers (TUNEL staining)

    • Functional assays relevant to the tissue being studied

What control experiments are essential when using SEMA6B antibodies in mechanistic studies?

To ensure scientific rigor when investigating SEMA6B function:

• Antibody specificity controls:

  • Isotype control: Use rabbit IgG at the same concentration as the SEMA6B antibody

  • Absorption control: Pre-incubate antibody with excess SEMA6B fusion protein

  • Genetic controls: Include SEMA6B knockout or knockdown samples

• Expression validation controls:

  • Paired comparisons: Always analyze matched normal vs. disease tissues

  • Multiple detection methods: Confirm protein expression using at least two independent techniques (e.g., western blot and IHC)

  • mRNA correlation: Verify that protein expression patterns match mRNA levels

• Functional assay controls:

  • Rescue experiments: Re-express SEMA6B in knockout cells to confirm specificity of observed phenotypes

  • Dose-dependency: Test effects across a range of SEMA6B expression levels

  • Time-course analysis: Document changes in inflammatory markers or apoptosis at multiple timepoints after SEMA6B manipulation

• In vivo controls for therapeutic targeting:

  • Include both positive controls (disease model without intervention) and negative controls (healthy animals)

  • Use scrambled siRNA as control for SEMA6B siRNA treatment

  • Monitor off-target effects by assessing related semaphorin family members

How should researchers interpret changes in SEMA6B expression in relation to disease progression?

When analyzing SEMA6B expression patterns:

• Prognostic value assessment:

  • In HBV-ACLF: SEMA6B shows diagnostic value with AUROC = 0.9788 in sequencing cohort and 0.9026 in validation cohort, with significantly higher levels in non-survivors

  • In colorectal cancer: High SEMA6B expression associates with poor progression-free survival, serving as an independent prognostic factor

• Correlation with disease markers:

  • For liver diseases: Connect SEMA6B levels with:

    • Liver function tests (ALT, AST, bilirubin)

    • Inflammatory markers (CRP, proinflammatory cytokines)

    • Hepatocyte damage markers

  • For cancer: Correlate SEMA6B expression with:

    • Tumor stage and grade

    • Immune cell infiltration levels

    • Immunosuppressive molecule expression

• Interpretation framework for expression data:

  • Consider tissue-specific contexts

  • Analyze expression in specific cell populations (e.g., macrophages vs. hepatocytes)

  • Account for temporal changes during disease progression

  • Integrate with clinical outcomes data

What are common technical challenges when working with SEMA6B antibodies and how can they be addressed?

Researchers may encounter these challenges:

• Background signal issues:

  • Problem: Non-specific binding in immunohistochemistry

  • Solution: Optimize blocking conditions (increase BSA concentration to 3-5%), extend blocking time, and titrate antibody dilutions within the recommended 1:20-1:200 range

• Signal detection sensitivity:

  • Problem: Weak signal in low-expressing samples

  • Solution: Consider signal amplification methods:

    • Use biotin-conjugated SEMA6B antibodies with streptavidin-HRP

    • Implement tyramide signal amplification for IHC/IF

    • Increase antibody concentration while monitoring background

• Epitope accessibility issues:

  • Problem: Masked epitopes in formalin-fixed tissues

  • Solution:

    • Optimize antigen retrieval methods (heat-induced vs. enzymatic)

    • Test multiple antibody clones that recognize different epitopes

    • Consider native vs. denatured protein detection methods

• Validation across species:

  • Problem: Limited cross-reactivity information

  • Solution:

    • Test antibody reactivity in multiple species

    • Include appropriate positive controls from target species

    • Use sequence homology analysis to predict cross-reactivity

How can researchers integrate SEMA6B antibody data with other molecular techniques for comprehensive pathway analysis?

A multi-modal approach enhances mechanistic insights:

• Integrative analysis workflow:

  • Start with antibody-based detection of SEMA6B protein levels

  • Correlate with mRNA expression by qRT-PCR or RNA-seq

  • Use phospho-specific antibodies to assess activation of downstream signaling pathways

  • Implement protein-protein interaction studies to identify binding partners

• Multi-omics data integration strategy:

  • Combine proteomics and transcriptomics:

    • Use LC-MS/MS to identify proteins interacting with immunoprecipitated SEMA6B

    • Correlate with RNA-seq data to identify gene expression changes

    • Example: Genes significantly associated with highly expressed SEMA6B were enriched in inflammation and apoptosis pathways in HBV-ACLF non-survivors

  • Integrate with genomics:

    • Analyze SNPs or mutations in SEMA6B that might affect antibody binding

    • Correlate genetic variations with protein expression patterns

• Pathway analysis frameworks:

  • After antibody-based quantification:

    • Use the ESTIMATE algorithm to calculate stromal scores, immune scores, and tumor purity

    • Correlate these metrics with SEMA6B expression levels

    • Implement functional enrichment analysis to identify biological processes associated with SEMA6B expression changes

  • Cell-type specific analysis:

    • Use FACS to isolate specific cell populations

    • Analyze SEMA6B expression in each population

    • Correlate with cell-type specific markers and functions

How can SEMA6B antibodies be utilized in therapeutic development research?

SEMA6B has emerged as a potential therapeutic target, particularly in liver diseases and cancer:

• Target validation experiments:

  • Use antibodies to confirm SEMA6B expression in target tissues

  • Correlate expression with disease severity

  • Document intracellular localization to inform drug design strategy

• Therapeutic antibody development pipeline:

  • Screen antibodies for functional blocking activity

  • Test effects on:

    • Inflammatory cytokine production

    • Cell proliferation, migration, and invasion

    • Apoptosis induction or prevention

  • Evaluate in both in vitro and in vivo models

• Therapeutic siRNA testing framework:

  • Use antibodies to confirm knockdown efficiency

  • Document therapeutic effects in disease models:

    • Improved liver function

    • Reduced inflammation

    • Decreased hepatocyte apoptosis

  • This approach has shown promise in liver failure mouse models

• Combination therapy assessment:

  • Use SEMA6B antibodies to monitor target engagement during combination treatments

  • Test SEMA6B inhibition alongside immune checkpoint inhibitors in cancer models

  • Evaluate synergistic effects with anti-inflammatory agents in liver disease models

What is the role of SEMA6B in immune cell infiltration and how can antibodies help investigate this phenomenon?

SEMA6B appears to significantly influence immune cell infiltration, particularly in cancer:

• Immunophenotyping protocol:

  • Use multicolor flow cytometry with SEMA6B antibodies

  • Co-stain for markers of:

    • CD4+ T cells

    • Macrophages

    • MDSCs

    • Tregs

    • Neutrophils

    • Dendritic cells

• Spatial distribution analysis:

  • Implement multiplex immunofluorescence using SEMA6B antibodies (1:20-1:200 dilution)

  • Map immune cell localization relative to SEMA6B-expressing cells

  • Quantify cell-cell distances and interaction frequencies

  • Correlate with clinical outcomes

• Mechanistic investigation approach:

  • Use antibodies to confirm SEMA6B expression in immune cells

  • Perform co-culture experiments between:

    • SEMA6B-overexpressing cancer cells and immune cells

    • SEMA6B-knockdown cancer cells and immune cells

  • Measure changes in immune cell recruitment, activation, and function

• Correlation analysis framework:

  • Calculate correlation coefficients between SEMA6B expression and:

    • Immune cell infiltration levels

    • Immune checkpoint molecule expression

    • Immunosuppressive cytokine production

  • Integrate findings from TIMER database analysis showing strong positive correlations with multiple immune cell types