OXCT1 Antibody

What is OXCT1 and why is it important in metabolic research?

OXCT1 is a mitochondrial matrix enzyme that plays a crucial role in ketone body metabolism. It catalyzes the transfer of CoA from succinyl-CoA to acetoacetate, generating acetoacetyl-CoA, which then enters the citric acid cycle . This process is particularly important during periods of glucose scarcity, such as fasting or prolonged exercise, when ketone bodies serve as alternative energy sources for tissues . The enzyme ensures that tissues efficiently convert ketone bodies like acetoacetate and beta-hydroxybutyrate into acetyl-CoA to fulfill energy requirements when carbohydrates are sparse .

Recent research has expanded our understanding of OXCT1 beyond its metabolic role, revealing its involvement in cancer progression, chemoresistance, and signaling pathways like NF-κB, making it a significant target for investigation in multiple fields .

What are the typical expression patterns of OXCT1 in different tissues?

OXCT1 exhibits a tissue-specific expression pattern that correlates with its metabolic function:

This expression pattern has been consistently observed across human, mouse, and rat samples . Interestingly, OXCT1 expression is often upregulated in various cancer types compared to corresponding normal tissues, including non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) .

What is the molecular weight and structure of OXCT1?

OXCT1 is a 520 amino acid protein with a calculated molecular weight of 56 kDa . In Western blot analyses, it typically appears as a band at approximately 56 kDa, though some antibodies may detect it between 52-56 kDa . The protein is encoded by the nuclear genome but functions in the mitochondrial matrix after appropriate trafficking and processing .

The functional enzyme exists as a dimer, and its activity depends on the transfer of CoA groups between substrates. OXCT1's structural features enable it to efficiently catalyze the reversible transfer of CoA from succinyl-CoA to acetoacetate in ketone body metabolism .

What are the most reliable applications for OXCT1 antibodies in research?

Based on validation data from multiple sources, OXCT1 antibodies demonstrate reliable performance in the following applications:

When selecting an antibody for experimental applications, researchers should consider the host species, clonality (monoclonal vs. polyclonal), and validated reactivity with their experimental model .

How can I validate the specificity of an OXCT1 antibody for my research?

Proper validation of antibody specificity is critical for reliable research results. For OXCT1 antibodies, consider these validation strategies:

• Knockout/Knockdown Controls: Use OXCT1 knockout cell lines (such as the OXCT1 knockout HeLa cell line) to confirm antibody specificity . Observe the disappearance of the target band in Western blot.

• Multiple Antibody Approach: Compare results from at least two independent antibodies raised against different epitopes of OXCT1.

• Tissue Expression Pattern: Verify that the antibody shows appropriate tissue expression patterns (strong in brain, heart, skeletal muscle; weak/absent in liver) .

• Band Size Verification: Confirm that the detected protein appears at the expected molecular weight (~56 kDa) .

• Recombinant Protein Control: Use purified recombinant OXCT1 as a positive control in Western blot applications .

These validation steps ensure that experimental findings are truly attributable to OXCT1 rather than non-specific binding or artifacts .

What are the optimal protocols for Western blot detection of OXCT1?

For optimal Western blot detection of OXCT1, follow these technical recommendations:

• Sample Preparation:

  • For tissue samples: Homogenize in RIPA buffer containing protease inhibitors

  • For cells: Lyse in buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, and protease inhibitors

• Gel Electrophoresis:

  • Load 20-30 μg of protein per lane

  • Use 10-12% SDS-PAGE gels for optimal resolution

• Transfer and Blocking:

  • Transfer to nitrocellulose membrane at 100V for 60-90 minutes

  • Block with 3-5% non-fat milk in TBS-T (0.1% Tween-20) for 1 hour at room temperature

• Antibody Incubation:

  • Primary antibody dilutions:

    • For polyclonal antibodies: 1:1000-1:2000

    • For monoclonal or recombinant antibodies: 1:5000-1:10000

  • Incubate overnight at 4°C

  • Secondary antibody: 1:5000-1:10000, incubate for 1 hour at room temperature

• Detection Controls:

  • Positive controls: HeLa, Jurkat, or A549 cell lysates; brain or heart tissue lysates

  • Negative controls: OXCT1 knockout cell lysates; liver tissue (low expression)

Following this protocol should yield a clear band at approximately 56 kDa representing OXCT1 protein .

How does OXCT1 contribute to cancer progression and chemoresistance?

Recent studies have revealed OXCT1's important roles in cancer beyond its metabolic function:

• Regulation of NF-κB Signaling:

  • OXCT1 activates the NF-κB signaling pathway in multiple cancer types

  • This activation promotes cancer cell survival, proliferation, and chemoresistance

  • Mechanistically, OXCT1 influences NF-κB activation through modulation of β-hydroxybutyrate (β-HB) levels

• Gemcitabine Resistance in Pancreatic Cancer:

  • High OXCT1 expression correlates with shorter relapse-free survival in gemcitabine-treated pancreatic cancer patients

  • OXCT1 overexpression inhibits apoptosis after gemcitabine treatment

  • OXCT1 knockdown increases sensitivity to gemcitabine therapy

  • This resistance mechanism operates through the NF-κB pathway

• Metabolic Reprogramming in Lung Cancer:

  • OXCT1 maintains β-hydroxybutyrate homeostasis, activating SREBP1

  • This leads to regulation of TRIM21 expression and subsequent effects on p65 stability

  • The pathway ultimately promotes tumor initiation and progression in non-small cell lung cancer

These findings suggest that OXCT1 could serve as both a biomarker and therapeutic target in cancer treatment, particularly for addressing chemoresistance .

What methods are available for studying OXCT1-mediated protein succinylation?

Recent research has identified OXCT1 as a potential succinyltransferase that can modify other proteins. For studying OXCT1-mediated protein succinylation:

• Western Blotting Approach:

  • Use anti-succinyllysine antibodies to detect global protein succinylation changes

  • Compare succinylation patterns in OXCT1 overexpression versus knockdown models

  • Perform co-immunoprecipitation to identify interaction partners

• Mass Spectrometry Detection:

  • Apply PASEF (Parallel Accumulation Serial Fragmentation) mass spectrometry to identify specific succinylation sites

  • Use site-directed mutagenesis to validate identified sites (e.g., LACTB K284)

• In Vitro Succinylation Assay:

  • Express and purify GST-tagged OXCT1 protein from E. coli

  • Perform in vitro succinylation reactions with potential substrate proteins

  • Detect succinylation using anti-succinyllysine antibodies or mass spectrometry

• Experimental Controls:

  • Use OXCT1 enzymatic mutants that lack transferase activity

  • Include known substrates like LACTB as positive controls

  • Verify results through multiple detection methods

This emerging field links OXCT1's enzymatic activity to protein post-translational modifications, potentially expanding our understanding of how metabolic enzymes regulate cellular signaling .

How can I differentiate between mitochondrial and potential non-mitochondrial functions of OXCT1?

Distinguishing between mitochondrial and potential non-mitochondrial functions of OXCT1 requires careful experimental design:

• Subcellular Fractionation:

  • Separate mitochondrial, cytosolic, and nuclear fractions using differential centrifugation

  • Analyze OXCT1 distribution across fractions by Western blotting

  • Use established markers for each fraction (e.g., COX4I1 for mitochondria, GAPDH for cytosol)

• Immunofluorescence Microscopy:

  • Perform co-localization studies with mitochondrial markers (e.g., MitoTracker)

  • Use high-resolution confocal microscopy to determine precise subcellular localization

  • Compare OXCT1 localization in different cell types and under various metabolic conditions

• Engineered OXCT1 Variants:

  • Create constructs with mutated mitochondrial targeting sequences

  • Express tagged versions that localize to specific compartments

  • Assess functional consequences of altered localization

• Functional Assays:

  • Measure ketone body metabolism in mitochondrial preparations

  • Assess non-canonical functions (e.g., protein succinylation) in different cellular compartments

  • Determine metabolite profiles (particularly β-hydroxybutyrate) in response to OXCT1 manipulation

Understanding the full spectrum of OXCT1's functions requires careful consideration of its potential activities outside its canonical mitochondrial role .

Why might I observe multiple bands when using OXCT1 antibodies in Western blot?

Multiple bands in OXCT1 Western blots can result from several factors:

• Protein Isoforms:

  • Some data suggests potential isoforms of OXCT1 (e.g., observed bands at 56 kDa and 86 kDa)

  • Verify with isoform-specific primers in RT-PCR to confirm expression of specific variants

• Post-translational Modifications:

  • OXCT1 may undergo modifications like phosphorylation or succinylation

  • These modifications can alter protein migration on gels

• Processing of Mitochondrial Targeting Sequence:

  • OXCT1 contains a mitochondrial targeting sequence that is cleaved upon import

  • Incomplete processing may result in multiple bands

• Non-specific Binding:

  • Some antibodies may cross-react with related proteins

  • Validate specificity using OXCT1 knockout samples

• Degradation Products:

  • Improper sample handling may lead to protein degradation

  • Use fresh samples with protease inhibitors

To resolve this issue, include appropriate controls such as OXCT1 knockout cell lysates, and optimize antibody dilution and blocking conditions .

What are the key considerations for immunohistochemical detection of OXCT1?

For optimal immunohistochemical detection of OXCT1 in tissue samples:

• Antigen Retrieval:

  • Recommended method: TE buffer at pH 9.0

  • Alternative: Citrate buffer at pH 6.0 if TE buffer yields high background

  • Heat-induced epitope retrieval using pressure cooker or microwave

• Antibody Selection and Dilution:

  • Polyclonal antibodies: 1:50-1:500 dilution

  • Monoclonal antibodies: 1:500-1:2000 dilution

  • Incubate overnight at 4°C for optimal signal-to-noise ratio

• Positive Control Tissues:

  • Brain, heart, and kidney tissues show strong OXCT1 expression

  • Testis shows strong cytoplasmic positivity in cells in seminiferous ducts

  • Heart tissue shows strong cytoplasmic positivity in cardiomyocytes

• Negative Control Tissues:

  • Liver typically shows minimal OXCT1 expression

  • Include antibody omission controls and isotype controls

• Interpretation of Results:

  • OXCT1 typically shows cytoplasmic localization with mitochondrial pattern

  • Expression patterns may vary by tissue and pathological state

  • Compare with normal adjacent tissue in cancer studies

Following these guidelines ensures reliable and reproducible detection of OXCT1 in tissue samples for both research and potential diagnostic applications .

How should I design experiments to study the role of OXCT1 in ketone metabolism in cancer cells?

To investigate OXCT1's role in ketone metabolism in cancer contexts:

• Cell Line Selection:

  • Use multiple cancer cell lines with varying OXCT1 expression levels

  • Include both glycolytic and oxidative phosphorylation-dependent lines

  • Common lines used: HeLa, A549, H1299, Jurkat, MCF-7

• Genetic Manipulation Approaches:

  • OXCT1 knockdown: Use validated siRNA or shRNA constructs

  • OXCT1 overexpression: Use expression vectors with appropriate tags

  • CRISPR-Cas9 knockout: Generate complete OXCT1 knockout models

• Metabolic Assays:

  • Measure uptake and utilization of β-hydroxybutyrate and acetoacetate

  • Determine acetyl-CoA levels and TCA cycle intermediates

  • Use isotope-labeled ketone bodies to trace metabolic flux

• Functional Measurements:

  • Assess cell proliferation under various nutrient conditions (glucose-rich, glucose-depleted, ketone-supplemented)

  • Measure oxygen consumption rate and extracellular acidification rate

  • Determine ATP production with and without ketone supplementation

• Signaling Pathway Analysis:

  • Investigate β-HB-SREBP1-TRIM21-p65 signaling axis

  • Assess NF-κB pathway activation in response to OXCT1 manipulation

  • Use pathway inhibitors to confirm mechanistic links

This comprehensive approach allows for detailed understanding of how OXCT1 and ketone metabolism influence cancer cell behavior, potentially revealing new therapeutic targets .

What new functions of OXCT1 beyond ketone metabolism have been recently discovered?

Recent research has uncovered several non-canonical functions of OXCT1:

• Protein Succinylation Activity:

  • OXCT1 can act as a succinyltransferase, promoting protein succinylation

  • LACTB has been identified as a specific target for OXCT1-mediated succinylation at K284

  • This represents a novel enzymatic activity distinct from its ketolytic role

• Signaling Pathway Regulation:

  • OXCT1 maintains β-hydroxybutyrate homeostasis, which functions as a signaling metabolite

  • β-HB activates SREBP1, connecting OXCT1 to transcriptional regulation

  • This pathway ultimately influences NF-κB signaling, linking metabolism to inflammation and cancer progression

• Chemoresistance Mechanisms:

  • OXCT1 promotes resistance to gemcitabine in pancreatic cancer through NF-κB pathway activation

  • This function appears independent of ketone body metabolism and more related to cell survival signaling

• Potential Epigenetic Regulation:

  • β-HB levels, regulated by OXCT1, may influence histone acetylation and gene expression

  • This suggests a potential role for OXCT1 in epigenetic regulation through metabolic intermediates

These discoveries highlight OXCT1 as a multifunctional protein that bridges metabolism with signaling, cell survival, and potentially epigenetic regulation .

How is OXCT1 expression regulated in normal and disease states?

The regulation of OXCT1 expression shows complex patterns in both normal physiology and disease:

• Physiological Regulation:

  • Upregulated during fasting and ketogenic diets to support ketone utilization

  • Tissue-specific expression (high in brain, heart, skeletal muscle; low in liver)

  • Developmentally regulated, with highest expression in tissues with high energy demands

• Cancer-Associated Dysregulation:

  • Significantly upregulated in multiple cancer types compared to adjacent normal tissues

  • In NSCLC, OXCT1 expression is higher than in normal lung epithelial cells

  • In PDAC, high OXCT1 expression correlates with lymph node metastasis and vessel invasion

• Prognostic Significance:

  • High OXCT1 expression correlates with worse survival in gemcitabine-treated pancreatic cancer patients

  • May serve as a prognostic biomarker for treatment response and patient outcomes

  • Analysis of 93 gemcitabine-treated PDAC patients showed significantly shorter relapse-free survival in high OXCT1 expression group (median RFS: 495 days versus 579 days)

• Potential Regulatory Mechanisms:

  • Transcriptional regulation by metabolic stress and nutrient availability

  • May be regulated by oncogenic signaling pathways

  • Post-translational modifications likely contribute to activity regulation

Understanding these regulatory mechanisms could provide insights for targeting OXCT1 in disease states while minimizing effects on normal physiological functions .

What are the most promising strategies for targeting OXCT1 in cancer therapy?

Based on recent findings, several strategies for targeting OXCT1 in cancer therapy show promise:

• Direct Enzymatic Inhibition:

  • Develop small-molecule inhibitors of OXCT1's ketolytic activity

  • Target the CoA transferase active site to block enzymatic function

  • Design structure-based inhibitors using crystal structure information

• Disruption of OXCT1-Mediated Signaling:

  • Target the β-HB-SREBP1-TRIM21-p65 signaling axis

  • Combine OXCT1 inhibition with NF-κB pathway inhibitors

  • Focus on disrupting OXCT1's role in chemoresistance mechanisms

• Metabolic Context Exploitation:

  • Combine OXCT1 inhibition with glycolysis inhibitors to target cancer metabolism

  • Utilize ketogenic diets to enhance dependency on OXCT1 in some tumors

  • Design metabolic synthetic lethality approaches

• RNA Interference and Novel Biologics:

  • Develop siRNA delivery systems targeting OXCT1 mRNA

  • Create antisense oligonucleotides to downregulate OXCT1 expression

  • Explore PROTAC approaches to induce OXCT1 protein degradation

• Biomarker-Guided Therapy:

  • Use OXCT1 expression as a biomarker for patient stratification

  • Focus on cancers with high OXCT1 dependency (e.g., PDAC, NSCLC)

  • Combine with other metabolic biomarkers for precision medicine approaches

These approaches could lead to novel therapeutic strategies for cancers that show dependence on OXCT1 for growth, survival, and chemoresistance .