TMC8 Antibody

What is TMC8 and why is it important in research?

TMC8, also known as EVER2 (Epidermodysplasia Verruciformis protein 2), is a transmembrane protein with a molecular weight of approximately 81.6 kDa in humans. It is located primarily in the endoplasmic reticulum and has been implicated in multiple biological processes, including immune response regulation, ion channel activity, and protection against certain viral infections, particularly human papillomavirus (HPV) .

TMC8 has gained research significance because:

• It forms part of a heterotrimeric complex with TMC6 (EVER1) and calcium and integrin-binding protein 1 (CIB1)

• Mutations in TMC8 are causally linked to Epidermodysplasia verruciformis (EV), a rare genetic disorder characterized by increased susceptibility to HPV infections

• Its expression has been associated with prognosis in multiple cancer types, including head and neck squamous cell carcinoma and hepatocellular carcinoma

What applications are TMC8 antibodies commonly used for?

TMC8 antibodies are utilized in various research applications with Western blot being the most widely used. According to technical specifications, the antibodies can be employed in:

When selecting a TMC8 antibody, researchers should consider the specific epitope recognition, as some antibodies are designed to target the N-terminal region (amino acids 50-79), which may influence detection capabilities depending on the protein conformation or interactions .

How should I design experiments to validate TMC8 antibody specificity?

Validating antibody specificity is crucial due to TMC8's structural similarity with other TMC family proteins and potential cross-reactivity. A comprehensive validation approach should include:

• Genetic controls: Use TMC8 knockout/knockdown samples as negative controls. Research has successfully employed Tmc8 Δ/Δ thymocytes as negative controls for antibody validation .

• Overexpression systems: Compare signals between wild-type cells and those transfected with TMC8-expressing constructs. Jurkat T cells have been effectively used for stable transduction or transfection of tagged TMC8 for antibody validation purposes .

• Cross-validation with multiple antibodies: Compare results using antibodies targeting different epitopes of TMC8.

• Western blot analysis: TMC8 typically appears as a doublet in thymocytes, with the upper band absent in Tmc6 Δ/Δ samples, providing a characteristic pattern for validation .

• Mass spectrometry confirmation: For definitive validation, immunoprecipitated proteins can be analyzed by mass spectrometry to confirm identity, as demonstrated in studies examining TMC6-TMC8-CIB1 complex formation .

What are the optimal conditions for detecting TMC8 in different cell types?

Detection efficiency varies significantly across cell types due to differential expression levels:

• T cells: Express TMC8 at high levels, making them ideal for antibody validation and protein interaction studies. TMC6 is expressed >6.6-fold higher in T cells than in keratinocytes .

• Keratinocytes: Express very low levels of TMC8, potentially requiring more sensitive detection methods .

• Cancer cells: Expression varies by cancer type; hepatocellular carcinoma shows upregulated TMC8 expression compared to adjacent tissues , while expression patterns differ in head and neck squamous cell carcinoma .

Recommended optimization steps:

• Adjust protein loading (20-50 μg total protein for Western blot)

• Modify antibody concentration (starting with 1:1000 dilution for Western blot)

• Extend primary antibody incubation time (overnight at 4°C often yields better results)

• Use enhanced chemiluminescence detection systems for low-abundance samples

How can I study the TMC6-TMC8-CIB1 protein complex?

The TMC6-TMC8-CIB1 heterotrimeric complex is critical for understanding TMC8 function. To effectively study this complex:

• Co-immunoprecipitation approach:

  • Use anti-TMC8 antibodies to pull down the complex

  • Include appropriate controls (Tmc6 Δ/Δ and Tmc8 Δ/Δ samples)

  • Detect interaction partners by Western blot with specific antibodies against TMC6 and CIB1

• Reciprocal co-immunoprecipitation:

  • Perform immunoprecipitation with anti-TMC6 or anti-CIB1 antibodies

  • Detect TMC8 in the precipitates to confirm interactions

• Tagged protein expression systems:

  • Express tagged versions (FLAG-TMC6 and HA-TMC8) in cell models

  • Use anti-tag antibodies for immunoprecipitation to avoid potential limitations of direct antibodies

Research has demonstrated that TMC6 and TMC8 heterodimerize and that these protein interactions are maintained even under CIB1 knockdown conditions, suggesting direct TMC6-TMC8 interaction independent of CIB1 .

What techniques can be used to visualize TMC8 subcellular localization?

To accurately determine TMC8 subcellular localization:

• Immunofluorescence microscopy:

  • Fix cells with 4% paraformaldehyde

  • Permeabilize with 0.1% Triton X-100

  • Block with 5% BSA or serum

  • Incubate with TMC8 antibody followed by fluorescent secondary antibody

  • Co-stain with organelle markers (e.g., calnexin for ER)

• Subcellular fractionation and Western blot:

  • Prepare cellular fractions (cytosolic, membrane, nuclear)

  • Compare TMC8 distribution across fractions

  • Include fraction-specific markers as controls

• Confocal microscopy with co-localization analysis:

  • Quantify co-localization with ER markers using Pearson's correlation coefficient

  • Compare wild-type and mutant TMC8 localization

TMC8 has been primarily detected in the cytoplasm, specifically in the endoplasmic reticulum, though interestingly, nuclear expression has been reported in some hepatocellular carcinoma cases, suggesting potential alternative functions that warrant further investigation .

How does TMC8 expression correlate with immune cell infiltration in tumors?

TMC8 expression shows significant correlations with immune cell infiltration in multiple cancer types:

• Head and neck squamous cell carcinoma (HNSC):

  • Positive correlation with CD4+ T cells and their subsets

  • Positive correlation with CD8+ T cells

  • Positive correlation with B cells and macrophages

  • TMC8 may play an anti-HPV role by regulating CD4+ T cells

• Renal cell carcinoma:

  • Positive correlation with B cells (TMC8 expression)

  • Positive correlation with CD8+ T cells

  • Positive correlation with NKT cells

These correlations suggest TMC8 may influence immune surveillance mechanisms within the tumor microenvironment. When investigating these associations, researchers should employ:

• Multiplex immunohistochemistry to simultaneously detect TMC8 and immune cell markers

• Deconvolution algorithms (TIMER, CIBERSORT, xCell) to estimate immune cell proportions from bulk RNA sequencing data

• Flow cytometry to directly quantify immune cell populations in relation to TMC8 expression

What are the contradictory findings regarding TMC8 expression in different cancer types?

The literature reveals interesting contradictions in TMC8's role across different cancer types:

These contradictions suggest context-dependent functions of TMC8 that may vary by:

• Tissue of origin

• Viral etiology (HPV-driven vs non-viral)

• Immune microenvironment composition

When investigating these discrepancies, researchers should consider:

• Using multiple detection methods (IHC, RT-qPCR, RNA-seq)

• Examining tissue-specific regulatory mechanisms

• Accounting for immune infiltration differences

• Analyzing mutation status alongside expression levels

How do TMC6 and TMC8 proteins cross-regulate each other?

Research has revealed a complex cross-regulatory relationship between TMC6 and TMC8 proteins:

• Post-translational regulation:

  • TMC6 band intensity decreases in Tmc8 Δ/Δ mice

  • The upper band of the TMC8 doublet is absent in Tmc6 Δ/Δ thymocytes

  • These changes occur at the protein level, as mRNA levels remain unchanged

• Heterodimer formation:

  • TMC6 and TMC8 form heterodimers

  • This heterodimer formation may protect against protein degradation

  • Similar to the anoctamin protein family structure

• Complex formation with CIB1:

  • Both TMC6 and TMC8 interact with CIB1

  • TMC6-TMC8 complex formation enhances the TMC8-CIB1 interaction

  • TMC6 and TMC8 interaction does not require CIB1

To study these regulatory relationships, researchers should consider:

• Using both single and double knockout models

• Employing proteasome inhibitors to assess degradation mechanisms

• Analyzing post-translational modifications that may affect protein stability

What are the key considerations when performing gene set enrichment analysis (GSEA) to identify TMC8-associated pathways?

When conducting GSEA to identify biological pathways associated with TMC8 expression:

• Sample stratification:

  • Divide samples based on TMC8 expression levels (using median expression as a threshold)

  • Create gene lists sorted according to expression differences between high and low TMC8 expression groups

• Reference dataset selection:

  • Consider multiple reference datasets (C2.CP and C5.BP MSigDB datasets are recommended)

  • Include immune-specific gene sets given TMC8's role in immune regulation

• Algorithm parameters:

  • Use appropriate statistical methods to determine whether reference gene set members are randomly distributed or enriched in the TMC8-high expression group

  • Apply suitable normalization methods for the expression data

• Validation strategies:

  • Confirm key pathways using alternative enrichment methods

  • Validate with protein-level assays for key pathway components

Previous GSEA analysis has shown TMC8 to be concentrated in multiple immune-associated signaling pathways, consistent with its role in immune cell regulation .

How can I design experiments to determine if TMC8 has ion channel functionality?

Testing TMC8's putative ion channel function requires specialized approaches:

• Electrophysiological methods:

  • Patch-clamp recording of cells overexpressing TMC8

  • Comparing wild-type vs mutant TMC8 variants

  • Analyzing ion selectivity and conductance properties

• Calcium imaging techniques:

  • Load cells with calcium-sensitive dyes or express genetically encoded calcium indicators

  • Measure calcium flux in response to stimuli in TMC8-expressing vs control cells

  • Research has shown that TMC8 limits the activation of Ca2+-dependent pathways

• Structural biology approaches:

  • Model TMC8 structure based on related ion channel proteins

  • Identify potential ion-conducting pores or domains

  • Create targeted mutations in these regions for functional testing

• Reconstitution in artificial membranes:

  • Purify TMC8 protein (potentially with TMC6)

  • Incorporate into lipid bilayers

  • Measure ion conductance directly

These experiments should include appropriate controls such as known ion channel inhibitors and TMC8 knockout/knockdown samples.

Why might TMC8 detection be inconsistent across different experimental systems?

Several factors can contribute to inconsistent TMC8 detection:

• Protein expression levels:

  • TMC8 is highly expressed in T cells but very low in keratinocytes

  • Expression in cancer cell lines is often below detection limits without enrichment

• Antibody specificity issues:

  • Commercial antibodies may lack specificity, as noted in research where custom antibodies were developed due to unreliable commercial options

  • Cross-reactivity with other TMC family members

• Post-translational modifications:

  • TMC8 undergoes glycosylation which can affect antibody recognition

  • The protein appears as a doublet in some tissues, with potential differential modification

• Complex formation requirements:

  • TMC8 stability may depend on TMC6 co-expression

  • Stable transfection/transduction approaches may be needed to achieve detectable levels

For reliable detection, researchers should:

• Use genetic controls (knockout/knockdown)

• Consider enrichment by immunoprecipitation before detection

• Validate antibodies against recombinant proteins

• Optimize protein extraction methods to preserve native conformation

What controls should be included when studying TMC8 in cancer tissues?

When examining TMC8 in cancer tissues, include these essential controls:

• Tissue-matched normal controls:

  • Adjacent non-tumorous tissue from the same patient

  • Normal tissue from healthy donors when possible

• Expression validation:

  • Complement IHC with RT-qPCR to confirm expression at mRNA level

  • Use multiple antibodies targeting different epitopes

• Cell type specificity controls:

  • Distinguish TMC8 expression in tumor cells from immune infiltrates

  • Include immunostaining for cell-type specific markers in consecutive sections

• Disease-specific controls:

  • Include HBV status for hepatocellular carcinoma studies

  • Include HPV status for head and neck cancer studies

• Technical controls:

  • Include isotype control antibodies

  • Use TMC8-deficient cell lines as negative controls

  • Include tissues known to express TMC8 (thymus, spleen) as positive controls

Research has demonstrated that careful pathological review is necessary to determine whether observed TMC8 expression is attributable to tumor cells or immune infiltrates .

What are the emerging research areas regarding TMC8's role in HPV immunity?

Several promising research directions are emerging:

• Mechanistic studies of T cell regulation:

  • Investigating how TMC8 regulates CD4+ T cell activity and differentiation

  • Examining its role in T cell memory formation against HPV

  • Understanding the mild T-cell abnormalities observed in TMC8-deficient individuals, including increases in memory, effector memory, and skin-homing T-cell subsets

• HPV life cycle regulation:

  • Exploring how TMC8 restricts HPV replication at the molecular level

  • Investigating interactions between TMC8 and viral proteins

• Signaling pathway analysis:

  • Further characterizing how TMC8 limits Ca2+-dependent pathway activation

  • Examining the relationship between TMC8 and the c-Jun N-terminal kinase/AP-1 signaling pathway, which may affect IL-6 expression

  • Investigating TMC8's role in NF-κB regulation

• Therapeutic targeting:

  • Developing approaches to modulate TMC8 function for enhanced anti-HPV immunity

  • Exploring TMC8 as a biomarker for immunotherapy response in HPV-associated cancers

How might single-cell analysis advance our understanding of TMC8 function?

Single-cell technologies offer promising approaches to resolve current contradictions in TMC8 research:

• Single-cell RNA sequencing:

  • Profiling TMC8 expression across heterogeneous cell populations within tissues

  • Identifying cell type-specific expression patterns

  • Correlating TMC8 expression with cell states and differentiation trajectories

• Single-cell proteomics:

  • Measuring TMC8 protein levels at single-cell resolution

  • Identifying co-expression patterns with interaction partners

• Spatial transcriptomics/proteomics:

  • Mapping TMC8 expression within tissue architecture

  • Correlating spatial expression patterns with pathological features

  • Examining TMC8 expression in relation to immune cell infiltration patterns

• CyTOF (mass cytometry):

  • Simultaneously measuring TMC8 with immune markers

  • Characterizing TMC8-expressing cells in the tumor microenvironment

These approaches could help resolve the apparent contradictions in TMC8's role across different cancer types and clarify its cell type-specific functions in normal physiology and disease.