TMEM173 Antibody

What is TMEM173 and what is its biological significance?

TMEM173 is the gene that encodes the protein STING (stimulator of interferon genes), a critical player in host defense against pathogens. STING is a four-transmembrane endoplasmic reticulum (ER) resident protein that exists as a homodimer. When activated by binding to cyclic dinucleotides (CDNs), STING undergoes a conformational change, then traffics through the Golgi to the perinuclear region where it activates TANK Binding Kinase 1 (TBK1), leading to type I interferon production .

STING is a key component in innate immunity and inflammation. Its importance is highlighted by the fact that mutations in the human TMEM173 gene cause a life-threatening auto-inflammatory disease called SAVI (STING-associated vasculopathy with onset in infancy). Furthermore, STING has emerged as a promising therapeutic target for both cancers and infectious diseases .

How does genetic variation in TMEM173 affect antibody selection and experimental design?

The human TMEM173 gene exhibits significant heterogeneity and population stratification, which researchers must consider when designing experiments and interpreting results:

Notably, R232/R232 is the dominant TMEM173 genotype in Europeans, while R232/HAQ is the most common genotype in East Asians. Approximately 30% of East Asians and 10% of Europeans have genotypes (HAQ/HAQ, HAQ/H232, or H232/H232) that may result in reduced STING function .

When selecting antibodies and designing experiments, researchers should consider:

• The specific epitope recognized by the antibody and whether it might be affected by genetic variants

• The genetic background of their experimental model or sample source

• The need for appropriate controls that account for genetic variation

What types of TMEM173/STING antibodies are available and how should I choose between them?

Several types of TMEM173/STING antibodies are available for research, each with specific advantages for different applications:

When choosing between these antibody types, consider:

• For polyclonal antibodies like AF6516, they recognize the region Ala215-Ser379 of human STING/TMEM173 (Accession # Q86WV6)

• For monoclonal antibodies like MAB7169, they offer high specificity for particular epitopes and are excellent for flow cytometry applications

• For complex applications requiring high specificity and reproducibility, monoclonal antibodies may be preferable

• For applications requiring detection of multiple epitopes or potentially modified forms, polyclonal antibodies may offer advantages

What are the recommended protocols for detecting TMEM173/STING via Western blot?

For optimal Western blot detection of TMEM173/STING:

• Sample preparation:

  • Use appropriate cell lysis buffers (compatible with membrane proteins)

  • Common cell lines expressing detectable levels include THP-1, U937, and peripheral blood lymphocytes

  • Load 0.2-1.0 mg/mL of protein lysate

• Electrophoresis conditions:

  • Use reducing conditions

  • For polyclonal antibody AF6516, use Immunoblot Buffer Group 8

  • For monoclonal antibody MAB7169, use Immunoblot Buffer Group 1

• Primary antibody incubation:

  • For AF6516: Use at 1 μg/mL concentration

  • For MAB7169: Use at 0.2 μg/mL concentration

• Detection system:

  • For AF6516: Use HRP-conjugated Anti-Sheep IgG Secondary Antibody (HAF016)

  • For MAB7169: Use HRP-conjugated Anti-Mouse IgG Secondary Antibody (HAF007)

• Expected results:

  • TMEM173/STING should be detected at approximately 40-42 kDa

  • Include proper positive controls (e.g., THP-1 cells) and negative controls (e.g., STING knockout cell lines)

How can I optimize flow cytometry protocols for TMEM173/STING detection?

For successful flow cytometry detection of TMEM173/STING:

• Cell preparation:

  • TMEM173/STING is primarily intracellular, requiring permeabilization

  • Fix cells with paraformaldehyde or Flow Cytometry Fixation Buffer (FC004)

  • Permeabilize with saponin or Flow Cytometry Permeabilization/Wash Buffer I (FC005)

• Antibody selection and staining:

  • Direct detection: Use PE-conjugated anti-TMEM173/STING (IC7169P)

  • Indirect detection: Use unconjugated primary (MAB7169) followed by fluorophore-conjugated secondary antibody

• Controls:

  • Include isotype controls (e.g., IC0041P for PE-conjugated antibodies)

  • Consider including STING-knockout cell lines as negative controls

• Cell types showing robust detection:

  • THP-1 human acute monocytic leukemia cells

  • U937 human histiocytic lymphoma cells

  • Human PBMC monocytes

• Gating strategy:

  • First gate on viable cells

  • Then gate on the cell population of interest

  • Compare staining to isotype control to identify positive population

How can TMEM173/STING antibodies be used to study the STING pathway in inflammatory diseases?

TMEM173/STING antibodies are valuable tools for investigating the role of STING in inflammatory diseases, particularly SAVI (STING-associated vasculopathy with onset in infancy):

• Pathway activation assessment:

  • Use antibodies against STING together with antibodies against phosphorylated TBK1 (pTBK1) and phosphorylated IRF3 (pIRF3) to monitor STING pathway activation

  • Western blot analysis can reveal increased pathway activation in disease states

• Immunohistochemistry applications:

  • Visualize STING expression in affected tissues

  • Co-stain with vascular markers (e.g., CD31) to examine associations between STING activation and vascular pathology

• Experimental manipulation:

  • Compare tissues/cells from control subjects versus patients with STING-associated diseases

  • Use STING antibodies to validate knockdown efficiency when using STING shRNA or other genetic approaches

  • Monitor pathway components after treatment with IFNAR-neutralizing antibodies or other therapeutics

• Vascular phenotype assessment:

  • Combine STING antibodies with vascular imaging techniques to correlate STING expression/activation with microvascular density, perfused capillary length, and vascular permeability

What role does TMEM173/STING play in cancer research and how can antibodies facilitate this work?

TMEM173/STING has emerged as a promising therapeutic target for cancer, and antibodies can help elucidate its role:

• STING-mediated immune surveillance:

  • Use TMEM173 antibodies to assess STING expression in tumor cells versus normal cells

  • Evaluate correlation between STING expression levels and clinical outcomes

• Cancer immunotherapy development:

  • Companies like Aduro Biotech and Novartis have invested over $250 million in developing STING-targeting cancer immunotherapies

  • TMEM173 antibodies can help validate target engagement of therapeutic compounds

• Pathway analysis in cancer models:

  • Use immunoprecipitation with TMEM173 antibodies to identify novel interaction partners in cancer cells

  • Combine with RNA-Seq data analysis to correlate STING activation with transcriptional changes

• Monitoring therapy response:

  • Flow cytometry with STING antibodies can track changes in STING expression/activation during treatment

  • Western blot analysis can reveal alterations in downstream signaling (pTBK1, pIRF3) following therapy

How do researchers investigate STING's role in innate immune signaling using TMEM173 antibodies?

TMEM173 antibodies enable detailed investigation of STING's role in innate immunity:

• Pathway activation analysis:

  • RNA-Seq data from HCT116 cells has shown that PARP1 depletion activates innate immune signaling

  • Gene Set Enrichment Analysis (GSEA) revealed "Interferon Alpha Response" as the top category of differentially expressed genes

  • Ingenuity Pathway Analysis (IPA) highlighted enrichment for Interferon-Stimulated Genes (ISGs)

  • TMEM173 antibodies can validate protein-level changes corresponding to transcriptional alterations

• RIG-I and MAVS dependency studies:

  • Western blotting with STING, RIG-I, MDA-5, TLR3, and MAVS antibodies can assess the efficiency of RNA silencing

  • These experiments help determine which components of the innate immune pathway are essential for ISG induction

  • qRT-PCR for ISGs like OAS1 and IFIT3 can quantify the functional consequences of silencing specific pathway components

• Imaging studies:

  • Immunofluorescence using TMEM173 antibodies can visualize STING localization within cells

  • In U937 human histiocytic lymphoma cells, STING staining localizes to the cytoplasm

  • This can be combined with other markers to track STING trafficking following activation

Why might I observe multiple bands when detecting TMEM173/STING by Western blot?

Multiple bands in TMEM173/STING Western blots can occur for several reasons:

• Post-translational modifications:

  • STING undergoes various modifications including phosphorylation and ubiquitination

  • Different modified forms may appear as distinct bands

• Splice variants:

  • Alternative splicing can generate different isoforms with varying molecular weights

• Protein degradation:

  • Partial degradation during sample preparation may produce fragments

  • Use fresh samples and protease inhibitors to minimize this issue

• Non-specific binding:

  • Some antibodies may show cross-reactivity with other proteins

  • In blots where multiple bands are observed, specific bands for STING are typically around 40-42 kDa

  • Non-specific bands are sometimes indicated with asterisks in reference blots

• Detection method considerations:

  • When using polyclonal antibodies like AF6516, confirm specificity by comparing with STING knockout cell lines

  • For monoclonal antibodies, verify that the observed band disappears in knockdown experiments

How can I validate TMEM173/STING antibody specificity for my experiments?

To ensure TMEM173/STING antibody specificity:

• Genetic validation approaches:

  • Compare staining/detection between:

    • Parental cell lines and STING knockout lines

    • Cells transfected with STING siRNA versus scramble controls

  • Simple Western analysis has shown specific STING detection at ~41 kDa in parental HeLa cells that is absent in STING knockout HeLa lines

• Immunoprecipitation validation:

  • Perform immunoprecipitation with anti-STING antibody followed by Western blot detection with a different anti-STING antibody

  • For example, Mouse Anti-Human STING/TMEM173 Monoclonal Antibody (MAB7169) has been validated for immunoprecipitation from PMA-treated THP-1 lysates

• Multi-antibody approach:

  • Use antibodies targeting different epitopes of STING

  • Consistent results across different antibodies increase confidence in specificity

• Control samples:

  • Include positive controls known to express STING (e.g., THP-1, U937 cells)

  • Use negative controls with low/no STING expression

  • Consider the impact of TMEM173 genetic variants on antibody binding

What methodological considerations are important when using TMEM173 antibodies for studying genetic variants?

When investigating TMEM173 genetic variants:

• Population-specific considerations:

  • Different populations have distinct distributions of TMEM173 alleles:

    • R232/R232 is dominant in Europeans

    • R232/HAQ is most common in East Asians

    • Approximately 30% of East Asians and 10% of Europeans have potentially loss-of-function genotypes

• Antibody selection based on variant regions:

  • Consider the specific epitope recognized by your antibody

  • For variants affecting antibody binding regions (e.g., R232H, R71H, G230A, R293Q), select antibodies targeting unaffected regions

• Experimental design:

  • Include appropriate controls representing different TMEM173 genotypes

  • Consider genotyping your experimental samples/cell lines

  • When using patient samples, account for potential genotype differences between cases and controls

• Functional validation:

  • Complement antibody-based detection with functional assays

  • Some variants (HAQ, H232) are likely loss-of-function alleles

  • Measuring downstream signaling (pTBK1, pIRF3) can help distinguish functional consequences of variants

How are TMEM173/STING antibodies being used to investigate vascular pathology?

Recent research has revealed STING's role in vascular remodeling and pathology:

• Stroke models and vascular analysis:

  • Researchers have examined STING expression in ischemic cortex after stroke

  • Immunoblot analysis of STING, pTBK1, and pIRF3 shows activation of this pathway post-stroke

  • STING knockdown using shRNA affects vascular responses after stroke

• Microvascular assessment techniques:

  • CD31-positive microvessel staining combined with STING antibodies can assess microvascular density

  • In-vivo multiphoton microscopy with intravenously injected FITC-dextran can evaluate:

    • Perfused capillary length

    • Vascular permeability

• Mechanistic insights:

  • STING-mediated effects on vascular remodeling appear to be related to neutrophil extracellular traps (NETs)

  • Immunoblot analysis of isolated neutrophils can examine STING pathway activation

  • IFNAR-neutralizing antibodies and STING shRNA can modulate these effects

• Quantitative assessment:

  • Confocal imaging and multiphoton microscopy enable quantification of:

    • Microvascular density in the peri-infarct cortex

    • Perfused capillary length

    • Permeability (P) product of FITC-dextran

  • These parameters show significant differences between control and STING-manipulated conditions

What considerations are important when designing TMEM173/STING antibody panels for comprehensive pathway analysis?

For comprehensive STING pathway analysis:

• Core pathway components to include:

  • STING/TMEM173: The central adaptor protein

  • TBK1 and phospho-TBK1: Downstream kinase activated by STING

  • IRF3 and phospho-IRF3: Transcription factor activated by TBK1

  • Type I interferons: End products of pathway activation

• Upstream regulators:

  • cGAS: DNA sensor that produces cGAMP, the endogenous STING ligand

  • RIG-I and MDA-5: RNA sensors that can activate STING-dependent signaling

  • MAVS: Mitochondrial adaptor that can signal to STING

• Panel optimization considerations:

  • Select antibodies with compatible species reactivity

  • Ensure antibodies work in your experimental system (cell types, tissues)

  • Validate antibody performance in multiple applications (Western blot, flow cytometry, IHC)

  • Include antibodies that can detect both total and activated (phosphorylated) forms of signaling proteins

• Application-specific panels:

  • For flow cytometry: Consider antibody conjugates compatible with your cytometer configuration

  • For multiplexed IHC: Select antibodies raised in different host species to avoid cross-reactivity

  • For Western blot: Ensure antibodies can detect denatured forms of the proteins