TRIM8 Antibody

What is TRIM8 and why is it important in research?

TRIM8, also known as RNF27 (RING finger protein, 27), is a member of the TRIM family proteins that functions at the crosstalk of cancer and innate immunity. TRIM8 can function in both nuclear and cytoplasmic compartments to regulate NF-κB, one of the central pathways in inflammatory responses . Research significance stems from its dual role as both tumor suppressor and oncogene depending on the cellular context. In breast cancer specifically, TRIM8 is downregulated compared to normal tissues, and lower expression correlates with worse prognosis, suggesting potential as a prognostic biomarker . Additionally, TRIM8 regulates estrogen receptor alpha (ERα) signaling in breast cancer, impacting cancer proliferation and tamoxifen sensitivity .

What are the key characteristics of TRIM8 antibodies used in research?

Commercial TRIM8 antibodies like the 27463-1-AP (polyclonal rabbit IgG) demonstrate reactivity with human samples with a calculated and observed molecular weight of 61 kDa . These antibodies are typically produced using TRIM8 fusion protein immunogens and purified using antigen affinity methods. For optimal results in research applications, TRIM8 antibodies are generally stored in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 and maintained at -20°C where they remain stable for approximately one year after shipment . The antibodies have been validated for multiple applications including Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), Immunoprecipitation (IP), and ELISA, with specific dilution recommendations for each technique (1:500-1:1000 for WB and 1:50-1:500 for IHC) .

How does TRIM8 expression vary across different tissue types and cancer models?

TRIM8 expression patterns show significant tissue variability, with altered expression in several cancer types compared to matched normal tissues. In breast cancer specifically, comprehensive analysis reveals that TRIM8 expression is dramatically lower in cancer tissues compared to normal breast tissue . In experimental models, TRIM8 has been studied extensively in ER-positive breast cancer cell lines including MCF7 and T47D, where its expression influences cell proliferation, migration, and response to treatments. Research demonstrates that the impact of TRIM8 is context-dependent - while it shows significant effects on ER-positive breast cancer cells, studies indicate it has limited impact on triple-negative breast cancer cells . When analyzing TRIM8 expression data, researchers should consider both protein and mRNA levels, as post-translational regulation may be important in different cellular contexts.

What are the optimal conditions for using TRIM8 antibodies in Western blotting?

For optimal Western blotting results with TRIM8 antibodies, researchers should:

• Prepare protein lysates using RIPA buffer to effectively extract TRIM8 from both nuclear and cytoplasmic compartments .

• Separate proteins using 10% SDS-PAGE gels, which provide appropriate resolution for the 61 kDa TRIM8 protein .

• Transfer proteins to 0.45 μm PVDF membranes for optimal binding.

• Block membranes with 5% skimmed milk in TBST for 2 hours at room temperature .

• Incubate with primary TRIM8 antibody at a dilution of 1:500-1:1000 overnight at 4°C .

• Wash thoroughly and incubate with appropriate secondary antibody (typically 1:5000) for 1 hour at room temperature .

• Perform detection using ECL immunoblotting kits with β-actin as a loading control .

Positive controls should include HepG2 or L02 cells, which have been validated to express detectable levels of TRIM8 . When analyzing results, the 61 kDa band should be observed, consistent with both the calculated and observed molecular weight of TRIM8 protein .

How should researchers optimize immunohistochemistry protocols for TRIM8 detection in tissue samples?

For effective immunohistochemical detection of TRIM8 in tissue samples:

• Perform antigen retrieval preferentially with TE buffer at pH 9.0, although citrate buffer at pH 6.0 can serve as an alternative .

• Use TRIM8 antibody at a dilution range of 1:50-1:500, with specific optimization recommended for each tissue type .

• Include positive control tissues such as human breast cancer tissue or human liver cancer tissue, which have been validated for TRIM8 detection .

• Employ appropriate blocking steps to minimize background staining.

• Optimize incubation times and temperatures based on signal intensity and background.

• Consider using detection systems with signal amplification for low-expressing samples.

When interpreting IHC results, researchers should evaluate both staining intensity and the percentage of positive cells. The subcellular localization information is particularly important for TRIM8, as it functions in both nuclear and cytoplasmic compartments, and differential localization may correlate with distinct functional states .

What experimental design considerations are important for TRIM8 knockdown and overexpression studies?

When designing TRIM8 knockdown or overexpression experiments:

For knockdown studies:

• Multiple siRNA sequences should be tested to ensure specificity and efficacy. Validated sequences include:

  • siRNA#1: 5'-GUGGACAACUGUUACUGUUTT-3'

  • siRNA#2: 5'-CAGACAAGCGCCUGGUGGATT-3'

  • siRNA#3: 5'-TGATAAGACGGAGGATGTCAGCTTCATGA-3'

• Transfection should be performed using appropriate reagents like Lipofectamine RNAiMAX following manufacturer's protocols .

• Verify knockdown efficiency at both protein level (Western blot) and mRNA level (qRT-PCR) .

For overexpression studies:

• Lentiviral-based systems have proven effective for stable TRIM8 overexpression .

• Include appropriate empty vector controls.

• Verify overexpression by Western blotting and qRT-PCR.

Functional assays to consider:

• Proliferation assays: CCK8 assay and plate cloning experiments

• DNA synthesis: EDU staining

• Migration: Wound-healing assay

• In vivo studies: Xenograft mouse models (using BALB/c nude mice supplemented with 17β-estradiol for ER-positive models)

When analyzing results, researchers should account for cell type-specific effects, as TRIM8 has shown differential effects in ER-positive versus triple-negative breast cancer cell lines .

How can researchers troubleshoot inconsistent results when detecting TRIM8 in different cellular compartments?

Inconsistent detection of TRIM8 across cellular compartments may stem from several methodological factors:

• Fractionation protocol efficiency: TRIM8 functions in both nuclear and cytoplasmic compartments, requiring efficient subcellular fractionation. Use validated markers like Histone H3 for nuclear fraction and β-Tubulin for cytoplasmic fraction to confirm separation quality .

• Fixation method impact: Different fixation protocols can affect epitope accessibility. For immunofluorescence studies detecting TRIM8, test multiple fixation methods (paraformaldehyde, methanol, or acetone) to determine optimal conditions.

• Antibody clone specificity: Different antibody clones may preferentially recognize distinct TRIM8 conformations or post-translationally modified forms. When inconsistencies arise, validate using multiple antibodies targeting different TRIM8 epitopes.

• Protein-protein interactions: TRIM8 interactions with binding partners may mask epitopes. Consider using multiple extraction conditions or detergents to disrupt protein complexes while maintaining TRIM8 structure.

• Cell type and context dependency: TRIM8 localization varies by cell type and cellular conditions. Experimental conditions should be standardized when comparing across cell lines, and positive controls specific to each compartment should be included.

For comprehensive analysis, consider complementing biochemical fractionation with imaging techniques to visualize TRIM8 distribution, and validate key findings using both approaches.

What potential conflicts might arise when interpreting TRIM8 function in different cancer models?

Several factors contribute to conflicting interpretations of TRIM8 function across cancer models:

• Dual role as tumor suppressor and oncogene: TRIM8 exhibits context-dependent functions, acting as a tumor suppressor in some cancers while showing oncogenic properties in others . This duality necessitates careful interpretation based on specific cellular contexts.

• p53 status dependency: TRIM8's effects on proteins like ΔNp63α appear to be p53-dependent. Research shows TRIM8 promotes ΔNp63α destabilization only in a functional p53 background . When interpreting TRIM8 function, researchers must consider and report p53 status of their models.

• Estrogen receptor status: In breast cancer models, TRIM8 function is significantly influenced by ERα status. TRIM8 knockdown promotes cell proliferation and reduces tamoxifen sensitivity in ER-positive cells but shows minimal effects in triple-negative cells .

• Immune microenvironment variations: TRIM8 expression correlates with immune characteristics, including negative correlations with CD4+ T cell, B cell, T follicular helper cell, and other immune cell populations . These correlations vary across cancer types and may influence interpretation of TRIM8 function.

• Methodological differences in protein detection: Differences in antibody specificity, detection methods, and subcellular fractionation protocols can lead to discrepancies in TRIM8 detection and subsequent functional interpretations.

To address these conflicts, researchers should clearly document experimental conditions, include comprehensive controls, and explicitly state the molecular context (p53 status, ER status, etc.) when reporting TRIM8 functions.

How can researchers reconcile contradictory data regarding TRIM8's role in cell proliferation?

When facing contradictory data about TRIM8's role in cell proliferation, consider these methodological approaches:

• Perform time-course analysis: TRIM8's effects may vary temporally. In MCF7 cells, TRIM8 overexpression arrests cells in G1 phase, while depletion increases cell viability and S-phase entry . Using synchronized cell populations and analyzing cell cycle distribution at multiple timepoints can provide more comprehensive insights.

• Evaluate multiple proliferation assays: Different assays measure distinct aspects of proliferation. Combining approaches such as:

  • MTT/CCK8 for metabolic activity

  • EDU staining for DNA synthesis

  • Plate cloning for long-term proliferative capacity

  • Cytofluorimetric analysis for cell cycle distribution
    can provide more nuanced understanding of TRIM8's effects.

• Assess molecular mechanisms in parallel: Simultaneously measure ΔNp63α and p53 levels when manipulating TRIM8, as TRIM8 inversely regulates these proteins which have opposing effects on proliferation .

• Conduct rescue experiments: To establish causality, perform molecular rescue experiments. In breast cancer cells, ERα knockdown restores cell proliferation and migration in TRIM8-depleted cells, confirming that TRIM8's anti-proliferative effects require ERα .

• Use multiple experimental models: TRIM8's effects may differ between in vitro and in vivo contexts. Xenograft models have confirmed that lentivirus-based TRIM8 overexpression inhibits breast tumor growth in vivo, supporting in vitro findings .

By implementing these approaches and clearly reporting cellular context, researchers can better reconcile seemingly contradictory data on TRIM8's proliferative effects.

How does TRIM8 regulate the stability of ΔNp63α, and what are the implications for p53 pathway research?

TRIM8 exhibits a complex regulatory relationship with ΔNp63α that has significant implications for p53 pathway research:

• Dual degradation mechanisms: TRIM8 promotes ΔNp63α destabilization through both Caspase 1-dependent and proteasomal pathways, but critically, this occurs only in cells with functional p53 . This p53-dependency creates an important regulatory circuit that links these related transcription factors.

• Molecular consequences: When TRIM8 levels are reduced, ΔNp63α stability increases, promoting cell proliferation and chemoresistance . This suggests TRIM8 functions as a molecular switch between pro-survival and pro-apoptotic pathways by simultaneously stabilizing p53 and destabilizing ΔNp63α.

• Experimental validation approaches: To study this mechanism, researchers should employ:

  • Cycloheximide chase experiments to quantify protein stability changes

  • Proteasome inhibitors (MG132) and caspase inhibitors to identify degradation pathways

  • Co-immunoprecipitation to detect direct interactions

  • Ubiquitination assays to assess post-translational modifications

• Physiological relevance: This regulatory mechanism appears particularly important in the cellular response to DNA damage, where TRIM8 may help coordinate appropriate cellular responses through the p53 family network .

• Research implications: Investigators studying p53 pathway function should consider TRIM8 expression levels as a potential confounding variable in their experimental systems, particularly when examining ΔNp63α-dependent phenotypes.

This sophisticated regulatory system underscores the importance of considering protein stability and degradation mechanisms when investigating transcription factor function in the p53 pathway.

What is the relationship between TRIM8 expression and immune cell populations in the tumor microenvironment?

TRIM8 demonstrates significant correlations with immune cell populations in the tumor microenvironment, particularly in breast cancer:

These findings position TRIM8 as a potential link between cancer cell-intrinsic properties and the tumor immune microenvironment, warranting further mechanistic investigation.

How does TRIM8 mechanistically regulate estrogen receptor signaling in breast cancer?

TRIM8 exerts significant regulatory effects on estrogen receptor signaling in breast cancer through several mechanisms:

• Regulation of ERα protein levels: TRIM8 knockdown significantly upregulates ERα protein levels in breast cancer cells, while TRIM8 overexpression reduces ERα protein levels . This regulation occurs regardless of estradiol (E2) treatment status.

• Modulation of ERα transcriptional activity: RNA sequencing analysis reveals that TRIM8 knockdown significantly increases ERα target gene expression (including GREB1, PS2, and CCND1) and activates estrogen signaling pathways . Reporter assays using ERE-luciferase constructs confirm that TRIM8 depletion enhances ERα transcriptional activity .

• Impact on tamoxifen sensitivity: TRIM8 knockdown reduces the sensitivity of MCF7 and T47D cells to tamoxifen, suggesting TRIM8 may influence endocrine therapy responses in ER-positive breast cancer .

• Functional dependency: Rescue experiments demonstrate that ERα knockdown can restore normal cell proliferation and migration in TRIM8-depleted cells, confirming that TRIM8's effects on these phenotypes require ERα .

• Research approaches for mechanism elucidation:

  • Protein-protein interaction studies to determine if TRIM8 directly associates with ERα

  • Ubiquitination assays to assess if TRIM8's E3 ligase activity targets ERα for degradation

  • ChIP-seq to examine if TRIM8 affects ERα chromatin binding patterns

  • Proteasome inhibition studies to determine the mechanism of ERα regulation

These findings suggest TRIM8 functions as a negative regulator of estrogen signaling in breast cancer, with potential implications for endocrine therapy resistance mechanisms.

What are the potential applications of TRIM8 as a prognostic biomarker in cancer?

TRIM8 shows considerable promise as a prognostic biomarker in cancer, particularly breast cancer:

• Expression-outcome correlations: Comprehensive analysis demonstrates that lower TRIM8 expression correlates with worse prognosis in breast cancer patients . This suggests potential utility as a prognostic indicator.

• ROC curve validation: Receiver operating characteristic (ROC) curve analysis has confirmed good performance in survival prediction, demonstrating the accuracy of TRIM8-based immune prognostic signatures .

• Nomogram integration: TRIM8 expression has been successfully integrated into prognostic nomograms, with calibration curves demonstrating model usefulness for predictions .

• Immune correlation advantages: TRIM8's correlations with immune cell populations provide additional prognostic information beyond simple expression levels, potentially enhancing its biomarker utility .

• Methodological considerations for clinical implementation:

  • Standardization of detection methods (IHC versus RNA-seq)

  • Establishment of clinically relevant expression thresholds

  • Validation across diverse patient populations

  • Integration with existing prognostic factors

  • Prospective clinical studies to confirm retrospective findings

• Therapy-specific prognostic value: Given TRIM8's influence on tamoxifen sensitivity , its expression might have particular value in predicting endocrine therapy responses in ER-positive breast cancer.

The multifaceted roles of TRIM8 in cancer biology, including its regulation of p53 pathway components, ERα signaling, and correlations with immune populations, suggest it may provide prognostic information that integrates multiple cancer hallmarks, potentially offering advantages over single-pathway biomarkers.

How can researchers design experiments to elucidate TRIM8's dual roles as tumor suppressor and oncogene?

To investigate TRIM8's context-dependent roles as both tumor suppressor and oncogene, researchers should consider these experimental design principles:

• Parallel model systems approach: Simultaneously study TRIM8 function in multiple cell lines representing:

  • Different cancer types (breast, colon, lung, etc.)

  • Different molecular subtypes within cancers (ER+/ER- breast cancer)

  • Different p53 status (wild-type, mutant, null)

  • Different stages of cancer progression

• Comprehensive functional readouts:

  • Proliferation: Short-term (MTT/CCK8) and long-term (colony formation) assays

  • Cell death: Apoptosis and other death modalities

  • Migration and invasion: Wound healing and transwell assays

  • In vivo tumorigenesis: Xenograft models with both gain and loss of function

• Molecular mechanism dissection:

  • Protein interactome analysis in different cellular contexts

  • ChIP-seq to identify context-dependent genomic binding sites

  • RNA-seq for global transcriptional effects

  • Subcellular localization analysis across conditions

• Signaling pathway integration:

  • Systematically investigate TRIM8's effect on canonical cancer pathways (p53, NF-κB, ERα)

  • Use pathway inhibitors to determine functional dependencies

  • Employ rescue experiments to establish causality

• Clinical correlation:

  • Analyze TRIM8 expression and mutation patterns across comprehensive cancer databases

  • Correlate with clinical outcomes in different cancer types

  • Assess associations with treatment responses

By implementing this systematic approach across diverse models and integrating findings with clinical data, researchers can develop a comprehensive understanding of the contextual factors that determine whether TRIM8 functions as a tumor suppressor or oncogene in specific cellular environments.

What novel methodologies might improve detection and functional analysis of TRIM8 in complex biological samples?

Emerging methodologies offer promising approaches to enhance TRIM8 detection and functional analysis:

• Proximity ligation assays (PLA): This technique can detect protein-protein interactions in situ with high sensitivity and specificity. For TRIM8, PLA could identify interactions with key binding partners like p53, ΔNp63α, or ERα in tissue samples, providing spatial context for TRIM8 function that traditional co-immunoprecipitation lacks.

• CRISPR-based functional screens: Pooled CRISPR screens in combination with TRIM8 manipulation can identify synthetic lethal or synthetic viable interactions, potentially revealing context-dependent functions. This approach could help explain why TRIM8 functions as a tumor suppressor in some contexts but an oncogene in others.

• Single-cell analysis techniques:

  • Single-cell RNA-seq to identify cell populations where TRIM8 expression correlates with specific transcriptional programs

  • Single-cell proteomics to assess TRIM8 protein levels and phosphorylation states at cellular resolution

  • Spatial transcriptomics to map TRIM8 expression patterns within tumor microenvironments in relation to immune infiltrates

• Advanced imaging approaches:

  • Super-resolution microscopy to visualize TRIM8 subcellular localization with nanometer precision

  • Live-cell imaging with fluorescently-tagged TRIM8 to track dynamic localization and protein interactions

  • Multiplex immunofluorescence to simultaneously detect TRIM8 and multiple markers in tissue samples

• Post-translational modification analysis:

  • Mass spectrometry approaches to comprehensively identify TRIM8 modifications

  • Site-specific antibodies against phosphorylated, ubiquitinated, or SUMOylated TRIM8

  • Functional studies using TRIM8 mutants lacking specific modification sites

• Organoid and patient-derived xenograft models:

  • These models better recapitulate in vivo tumor complexity compared to cell lines

  • Allow testing of TRIM8 manipulation in more physiologically relevant contexts

  • Can incorporate immune components to study TRIM8-immune interactions

Implementation of these advanced methodologies would significantly enhance our understanding of TRIM8 function in complex biological systems and potentially reveal novel therapeutic opportunities.

What are the key considerations for researchers beginning work with TRIM8 antibodies?

Researchers initiating studies with TRIM8 antibodies should consider these critical factors for successful experiments:

• Experimental validation: Always validate antibody specificity in your experimental system using positive controls (HepG2 or L02 cells) and negative controls (TRIM8 knockdown cells) . Western blot should show a band at the expected 61 kDa molecular weight .

• Application-specific optimization: Each application requires specific optimization:

  • WB: 1:500-1:1000 dilution with appropriate extraction buffers

  • IHC: 1:50-1:500 dilution with TE buffer pH 9.0 for antigen retrieval

  • IF: Optimize fixation methods based on cell type

• Cellular context awareness: TRIM8 function is highly context-dependent, influenced by:

  • p53 status (wild-type versus mutant)

  • ER status (positive versus negative)

  • Cancer type and tissue origin
    Document these contextual factors in experimental design and reporting.

• Subcellular localization considerations: TRIM8 functions in both nuclear and cytoplasmic compartments . Subcellular fractionation and localization studies can provide important functional insights.

• Functional readouts: When manipulating TRIM8, assess multiple functional outcomes:

  • Cell proliferation and cell cycle distribution

  • Protein stability of known targets (p53, ΔNp63α, ERα)

  • Pathway activation (NF-κB, estrogen signaling)

  • Immune cell correlations in complex samples

By carefully considering these factors in experimental design, researchers can generate more robust and interpretable data on TRIM8 function while avoiding common technical pitfalls associated with antibody-based research.

How might TRIM8 research evolve to address emerging challenges in cancer biology and therapy?

TRIM8 research is poised to address several emerging challenges in cancer biology:

• Therapy resistance mechanisms: Given TRIM8's role in regulating ERα and influencing tamoxifen sensitivity , further research could elucidate its contribution to endocrine therapy resistance in breast cancer. This might lead to TRIM8-based biomarkers for therapy selection or novel therapeutic approaches targeting TRIM8-dependent pathways.

• Immuno-oncology connections: The negative correlations between TRIM8 and various immune cell populations suggest TRIM8 may influence the tumor immune microenvironment. Future research should explore whether TRIM8 manipulation could enhance immunotherapy responses by modulating immune infiltration or function.

• Precision oncology applications: The context-dependent functions of TRIM8 underscore the need for precision medicine approaches. Developing diagnostic assays that integrate TRIM8 with other biomarkers could improve patient stratification for targeted therapies.

• Systems biology approaches: TRIM8's multiple roles in cancer, immunity, and cell signaling make it an ideal candidate for systems biology approaches that integrate multi-omics data to comprehensively map its functional network across cancer types and molecular contexts.

• Therapeutic targeting possibilities:

  • Small molecule modulators of TRIM8 expression or activity

  • Peptide inhibitors of specific TRIM8 protein interactions

  • Targeted degradation approaches (PROTACs) for context-specific TRIM8 modulation