ACOT11 Antibody

What is ACOT11 and what is its primary biological function?

ACOT11 (Acyl-CoA thioesterase 11) is an enzyme that hydrolyzes fatty acyl-CoA esters into free fatty acids and CoA. It demonstrates specific activity towards medium (C12) and long-chain (C18) fatty acyl-CoA substrates . ACOT11 is also known by several other names including Brown fat-inducible thioesterase (BFIT), Adipose-associated thioesterase, THEA, THEM1, and STARD14 .

While ACOT11's primary role involves fatty acid metabolism, its biological functions extend beyond this pathway. In mice, ACOT11 expression in brown adipose tissue is induced by cold exposure and suppressed by warmth, suggesting a role in thermogenesis . Additionally, studies have found higher ACOT11 expression in obesity-resistant mice compared to obesity-prone mice, indicating its potential involvement in energy homeostasis and metabolic regulation .

What are the typical tissue expression patterns of ACOT11?

ACOT11 demonstrates distinct tissue-specific expression patterns that researchers should consider when designing experiments:

• In mice, ACOT11 is primarily detected in brown adipose tissue (BAT)

• Using validated antibodies in knockout tissue models, ACOT11 has been observed in the high-speed pellet fraction of BAT preparations

• Notably, ACOT11 is generally not detected in high-speed pellet fractions from heart, skeletal muscle, liver, or kidney tissues

• In human cancer studies, ACOT11 shows tissue-specific expression alterations, with significant downregulation observed in clear cell renal cell carcinoma (ccRCC) compared to matched normal kidney tissues

This tissue-specific distribution makes ACOT11 an interesting target for studies of both metabolic regulation and disease-specific alterations.

What types of ACOT11 antibodies are available for research applications?

Several types of ACOT11 antibodies have been developed for research applications, each with specific characteristics:

When selecting an ACOT11 antibody, researchers should consider their specific application requirements, target species reactivity, and the protein region of interest. Many antibodies have been validated in knockout tissue models, which provides critical confidence in their specificity .

What are the recommended applications for ACOT11 antibodies?

ACOT11 antibodies have been validated for multiple research applications:

• Western Blotting: Most ACOT11 antibodies perform well in Western blot applications with recommended dilution ranges of 1:500 to 1:2,000. This technique allows detection of ACOT11 protein expression levels and can be used to verify knockdown efficiency in functional studies .

• Immunohistochemistry (IHC): ACOT11 antibodies are effective for tissue localization studies with recommended dilutions of 1:50 to 1:100. IHC has been successfully employed to analyze ACOT11 expression in clinical samples from cancer patients .

• ELISA/RIA: Several ACOT11 antibodies are specifically validated for enzyme immunoassays, providing quantitative measurements of ACOT11 levels .

• Flow Cytometry: Selected antibodies have been validated for flow cytometric applications, enabling cellular analysis of ACOT11 expression .

• Co-immunoprecipitation: ACOT11 antibodies have been successfully used in co-IP experiments to identify protein interaction partners, revealing binding with proteins such as CSE1L, which may contribute to oncogenic functions in lung cancer .

How should ACOT11 antibodies be validated for research use?

Proper validation of ACOT11 antibodies is critical for ensuring experimental reliability. Based on published research methodologies, the following validation approaches are recommended:

• Knockout/Knockdown Verification: Several studies have validated ACOT11 antibodies using tissues from knockout mice or cells with ACOT11 knockdown. This approach confirms antibody specificity by demonstrating absence of signal in knockout/knockdown samples .

• Recombinant Protein Controls: Testing antibodies against recombinant ACOT11 protein can verify binding specificity. Studies have used human ACOT11 (aa 19-250) expressed in E. coli as a positive control .

• Multiple Antibody Comparison: Using multiple antibodies targeting different epitopes of ACOT11 can help confirm specificity of detection .

• Western Blot Molecular Weight Verification: Confirming that the detected protein appears at the expected molecular weight (~50-55 kDa for full-length ACOT11) provides additional validation .

• Cross-Reactivity Testing: When using antibodies across species, cross-reactivity should be carefully assessed, particularly when extrapolating findings between mouse models and human samples .

A comprehensive validation approach combining these methods increases confidence in experimental results and minimizes the risk of artifacts or non-specific signals.

What roles does ACOT11 play in cancer progression and what are the implications for diagnostic applications?

ACOT11 exhibits complex and tissue-specific roles in cancer biology:

In lung cancer:

• High expression of ACOT11 correlates with poor prognosis in lung squamous carcinoma (LUSC) patients

• Functional studies show that ACOT11 knockdown inhibits cell proliferation, migration, and invasion both in vitro and in vivo

• ACOT11 knockdown promotes apoptosis and cell cycle arrest via multiple signaling pathways

• ACOT11 binds with CSE1L, an established oncogene in lung cancer, suggesting a potential molecular mechanism for its cancer-promoting effects

In renal cell carcinoma:

• Contrary to lung cancer, ACOT11 is significantly downregulated in clear cell renal cell carcinoma (ccRCC) compared to normal kidney tissues

• ACOT11 downregulation is observed in almost every matched normal-tumor pair, suggesting remarkable consistency

• ROC analysis reveals ACOT11 has extremely high diagnostic value for ccRCC with an AUC score of 0.964

• This expression pattern has been verified at both mRNA and protein levels in cell lines and clinical samples

These contrasting expression patterns in different cancer types suggest that ACOT11 may have context-dependent roles in cancer biology. The exceptional diagnostic performance of ACOT11 in ccRCC makes it a promising biomarker candidate for this cancer type, while its oncogenic properties in lung cancer suggest potential therapeutic targeting opportunities.

What methodological approaches are most effective for studying ACOT11 in cancer models?

Research into ACOT11's role in cancer has employed several effective methodological approaches:

• Gene Expression Modulation:

  • RNA interference via lentiviral vectors carrying shRNAs has been successfully used to knockdown ACOT11 expression in cancer cell lines

  • Stable cell lines with ACOT11 knockdown can be established using puromycin selection

  • The knockdown efficiency should be verified at both mRNA and protein levels using qRT-PCR and Western blotting

• Functional Assays:

  • Cell proliferation: High-content screening using fluorescent imaging cytometry provides quantitative assessment of cell growth over time

  • Colony formation: Assessing the ability of single cells to grow into colonies after ACOT11 knockdown reveals effects on clonogenic potential

  • Migration and invasion: These aspects can be examined using established assays to understand ACOT11's role in cancer metastasis

• In Vivo Models:

  • Female BALB/c nude mice have been used as in vivo models to study tumor growth following ACOT11 knockdown

  • This model allows assessment of ACOT11's effects on tumor development in a physiological context

• Molecular Mechanism Elucidation:

  • Transcriptional profiling: Microarray analysis following ACOT11 knockdown has revealed hundreds of differentially expressed genes (214 up-regulated and 397 down-regulated), providing insights into downstream mechanisms

  • Immunoprecipitation-mass spectrometry: This approach has identified 573 proteins that interact with ACOT11, offering a comprehensive interactome resource

  • Co-immunoprecipitation: This technique has confirmed specific interactions, such as the binding between ACOT11 and CSE1L

• Clinical Correlation:

  • Analysis of clinical data from resources like TCGA enables correlation of ACOT11 expression with patient survival and other clinical parameters

  • Immunohistochemistry on tissue microarrays allows assessment of protein expression in patient samples

Combining these approaches provides a comprehensive understanding of ACOT11's roles in cancer biology and its potential as a biomarker or therapeutic target.

How can ACOT11 expression be accurately quantified in experimental and clinical samples?

Accurate quantification of ACOT11 expression is essential for research and potential diagnostic applications. Several complementary methods have proven effective:

• Transcriptional Analysis:

  • Quantitative real-time PCR (qRT-PCR) has been successfully employed to measure ACOT11 mRNA levels in cell lines and clinical samples

  • For normalization, stable reference genes should be carefully selected based on the specific tissue or experimental context

  • Analysis of public transcriptomic datasets (e.g., TCGA, GEO) provides valuable insights into ACOT11 expression patterns across large patient cohorts

• Protein Detection and Quantification:

  • Western blotting allows semi-quantitative assessment of ACOT11 protein levels using validated antibodies with recommended dilutions of 1:500-2,000

  • Immunohistochemistry provides spatial information about ACOT11 expression in tissue sections, with optimal antibody dilutions between 1:50-100

  • Immunohistochemical scoring by experienced pathologists can quantify expression differences between normal and diseased tissues

• Receiver Operating Characteristic (ROC) Analysis:

  • ROC analysis has proven extremely valuable for assessing ACOT11's diagnostic potential, particularly in ccRCC where it achieved an AUC score of 0.964

  • This approach helps determine optimal cutoff values for distinguishing between normal and cancerous tissues

• Tissue Microarray (TMA) Analysis:

  • TMAs containing multiple patient samples enable high-throughput analysis of ACOT11 expression

  • This approach is particularly valuable for correlating expression with clinical parameters and outcomes

• Cell Line Validation:

  • Comparing ACOT11 expression across different cell lines helps identify suitable models for functional studies

  • Cell lines with moderate ACOT11 expression (e.g., A549 and NCI-H1975) are particularly useful as they allow both knockdown and overexpression experiments

When designing quantification experiments, researchers should consider tissue-specific expression patterns, as ACOT11 shows distinct expression profiles across different tissues and disease states.

What are the challenges in studying ACOT11 localization and how can they be overcome?

Determining the precise subcellular localization of ACOT11 presents several challenges that researchers should address:

• Prediction Algorithm Limitations:

  • Mitochondrial targeting sequence (MTS) prediction algorithms have high false negative rates (38-60%) and variable false positive rates (1-14%)

  • Many genuine mitochondrial proteins, including ACOT11, may have MTS predictions <30%, making computational predictions alone unreliable

• Experimental Approaches for Localization Studies:

  • Subcellular fractionation combined with Western blotting has successfully detected ACOT11 in the high-speed pellet fraction of BAT but not in other tissues

  • Protease protection assays can determine which compartment contains ACOT11 by selectively permeabilizing cellular membranes

  • Immunofluorescence microscopy with co-staining for compartment-specific markers provides spatial information about ACOT11 localization

• Antibody Validation for Localization Studies:

  • Use of antibodies validated in knockout tissues is crucial to ensure specificity in localization studies

  • Testing antibodies in multiple experimental contexts (Western blot, IHC, immunofluorescence) helps confirm consistent localization results

• Tissue-Specific Expression Considerations:

  • ACOT11's restricted expression pattern (primarily detected in BAT) means that careful tissue selection is critical for localization studies

  • When studying human samples, researchers should consider potential differences in tissue distribution compared to mouse models

• Overcoming Technical Challenges:

  • Combining multiple methodological approaches provides more robust evidence of localization

  • Using epitope-tagged ACOT11 constructs for overexpression studies can help overcome limitations of antibody detection sensitivity

  • Employing subcellular fractionation coupled with mass spectrometry offers an antibody-independent approach to localization

By addressing these challenges through rigorous experimental design and appropriate controls, researchers can accurately determine ACOT11's subcellular localization, which is important for understanding its function in normal and disease states.

How does ACOT11 interact with other proteins and signaling pathways in disease contexts?

Understanding ACOT11's protein interactions and pathway involvement is crucial for elucidating its role in disease:

• Identified Protein Interactions:

  • Immunoprecipitation-mass spectrometry has identified 573 proteins that interact with ACOT11, providing a comprehensive interactome map

  • Co-immunoprecipitation experiments have confirmed specific interactions, such as binding between ACOT11 and CSE1L in lung cancer

  • Other potential interaction partners investigated include CAMK2D, AHCY, EZR, RHEB, SMAD3, SQSTM1, ATP2A2, and CTNNB1

• Pathway Analysis:

  • Transcriptional profiling following ACOT11 knockdown revealed 214 up-regulated and 397 down-regulated genes, indicating involvement in multiple signaling networks

  • ACOT11 knockdown affects cell proliferation, migration, invasion, apoptosis, and cell cycle progression through these pathways

  • In lung cancer, ACOT11 appears to function as an oncogene, promoting tumor growth and invasion

• Tissue-Specific Interactions:

  • ACOT11's interactions may differ between tissues, as suggested by its contrasting roles in different cancer types

  • In ccRCC, ACOT11 is consistently downregulated, suggesting a potential tumor suppressor role in this context

  • In lung cancer, ACOT11 is highly expressed and associated with poor prognosis, indicating an oncogenic function

• Methodological Approaches to Study Interactions:

  • Flag-tagged ACOT11 overexpression coupled with co-immunoprecipitation and Western blotting allows verification of specific interactions

  • Functional validation through knockdown or overexpression of both ACOT11 and its interaction partners helps establish the biological significance of these interactions

  • Pathway analysis software applied to transcriptomic data can identify signaling networks affected by ACOT11 modulation

• Translational Implications:

  • Understanding ACOT11's interaction network provides potential targets for therapeutic intervention

  • The binding between ACOT11 and CSE1L, a known oncogene in lung cancer, suggests that disrupting this interaction might have therapeutic value

  • ACOT11's involvement in multiple signaling pathways indicates that its targeting might affect several cancer hallmarks simultaneously

Further investigation of these interactions using techniques like CRISPR-Cas9-mediated gene editing and high-resolution structural studies will advance our understanding of ACOT11's role in disease pathogenesis.

Future Research Directions and Opportunities

The current landscape of ACOT11 research points to several promising future directions:

• Diagnostic Biomarker Development:

  • The exceptional diagnostic value of ACOT11 in ccRCC (AUC=0.964) warrants further validation in larger, multicenter cohorts

  • Development of standardized assays for ACOT11 detection in clinical samples could accelerate its translation into diagnostic practice

  • Exploration of ACOT11's biomarker potential in other cancer types beyond lung and kidney cancer may reveal additional clinical applications

• Therapeutic Target Exploration:

  • ACOT11's role in promoting lung cancer progression suggests it could be a valuable therapeutic target

  • The interaction between ACOT11 and CSE1L provides a specific molecular mechanism that could be targeted for intervention

  • Development of small molecule inhibitors or peptide-based approaches to disrupt ACOT11's oncogenic functions represents an important research opportunity

• Mechanistic Studies:

  • Further characterization of the 573 proteins identified in ACOT11's interactome will deepen our understanding of its molecular functions

  • Investigation of tissue-specific regulatory mechanisms explaining ACOT11's contrasting roles in different cancer types will provide valuable insights

  • Exploration of ACOT11's role in metabolic regulation and its connection to cancer metabolism represents an intriguing research avenue

• Technical Advancements:

  • Development of more specific and sensitive antibodies against different ACOT11 epitopes will enhance detection capabilities

  • Application of advanced techniques like spatial transcriptomics and proteomics will provide higher-resolution insights into ACOT11's tissue-specific functions

  • Integration of multi-omics approaches will offer a more comprehensive understanding of ACOT11's role in health and disease