slc25a47b Antibody

What is SLC25A47 and why is it important in research?

SLC25A47 (Solute Carrier Family 25 Member 47) is a mitochondrial carrier protein primarily expressed in the liver. It has a reported length of 308 amino acid residues and a mass of 33.4 kDa in humans . This protein has gained research interest due to its potential role in energy metabolism and its liver-specific expression pattern. Initially described as a possible uncoupling protein that may catalyze the physiological "proton leak" in liver, more recent research has challenged this function . SLC25A47 is also known by several synonyms including hepatocellular carcinoma down-regulated mitochondrial carrier protein (HDMCP) .

The protein's research significance stems from its potential involvement in:

• Mitochondrial transport processes

• Liver-specific metabolic regulation

• Energy homeostasis

• Potential associations with metabolic disorders

What are the expression patterns of SLC25A47 in different tissues?

SLC25A47 demonstrates highly tissue-specific expression, being predominantly expressed in the liver in both humans and mice . This liver-specific expression pattern suggests a specialized function related to hepatic metabolism. Research indicates that SLC25A47 is not expressed in cancer cell lines, including those in the Cancer Cell Line Encyclopedia, which further supports its specialized role in differentiated liver tissue .

Expression data demonstrates:

• High expression in liver tissue

• Minimal to no expression in other tissues

• Absence in most established cancer cell lines

• Conservation of liver-specific expression across species (human, mouse)

What validated applications are available for SLC25A47 antibodies?

According to the search results, commercially available SLC25A47 antibodies have been validated for multiple applications. The Proteintech SLC25A47 rabbit polyclonal antibody (26292-1-AP) has been validated for the following applications:

Additionally, the Novus Biologicals goat polyclonal antibody has been validated for:

• Immunohistochemistry (2.5 μg/ml)

• Immunohistochemistry-Paraffin (2.5 μg/ml)

• Peptide ELISA (detection limit 1:16000)

These antibodies have demonstrated reactivity with human and mouse samples, making them suitable for comparative studies across these species .

What are the optimal sample preparation methods for detecting SLC25A47 in mitochondrial fractions?

For optimal detection of SLC25A47 in mitochondrial fractions, researchers should consider the following methodological approaches:

• Mitochondrial isolation: Since SLC25A47 is localized to mitochondria, proper mitochondrial isolation is critical. Differential centrifugation techniques should be employed to obtain enriched mitochondrial fractions.

• Buffer considerations: For immunohistochemistry applications with the Proteintech antibody, researchers should use TE buffer pH 9.0 for antigen retrieval, though citrate buffer pH 6.0 can be used as an alternative .

• Sample handling: Since the protein is stored in the mitochondrial membrane, care should be taken to avoid denaturation during preparation. For the Proteintech antibody, storage at -20°C is recommended, and the antibody is reported stable for one year after shipment .

• Co-localization studies: When performing immunofluorescence studies, Mitotracker Red FM has been successfully used as a mitochondrial matrix marker to confirm mitochondrial localization of SLC25A47 .

How can I effectively design experiments to investigate SLC25A47's role in mitochondrial transport?

To effectively investigate SLC25A47's role in mitochondrial transport, consider the following experimental approach based on published research:

• Subcellular localization confirmation:

  • Perform immunofluorescence with SLC25A47 antibodies and mitochondrial markers like Mitotracker Red FM

  • Conduct subcellular fractionation followed by Western blotting to confirm mitochondrial enrichment

• Functional assays:

  • High-resolution respirometry using platforms like Oroboros Oxygraph 2k

  • Implement Coupling Control Protocols to assess various respiratory states

  • Compare wild-type and SLC25A47-deficient or overexpressing systems

• Metabolomic analysis:

  • Assess impact on TCA cycle intermediates and amino acid metabolism

  • Measure acylcarnitine levels to assess fatty acid metabolism

  • Evaluate both mitochondrial and plasma metabolites

• Gene expression analysis:

  • Investigate regulation by PPARα and related metabolic pathways

  • Use qPCR to assess expression of related genes such as CPT2, ACADL, SLC25A20, and TFAM

The research by Peeters et al. found no significant uncoupling effect when implementing these methodologies, which contradicts earlier hypotheses about SLC25A47 function .

What are the recommended controls for validating SLC25A47 antibody specificity?

To ensure antibody specificity when studying SLC25A47, researchers should implement the following controls:

• Genetic validation:

  • Compare staining between wild-type and SLC25A47 knockout tissues/cells

  • Use SLC25A47-transfected cells (e.g., Hepa 1-6 cells which naturally lack SLC25A47 expression) versus control-transfected cells

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide to block specific binding

  • The Novus Biologicals antibody was raised against peptide sequence C-RYGNPDAKPTKAD, which could be used for competition studies

• Multiple antibody validation:

  • Compare results using antibodies targeting different epitopes of SLC25A47

  • For example, compare results between Proteintech's antibody (targeting SLC25A47 fusion protein Ag23376) and Novus Biologicals' antibody (targeting the internal region RYGNPDAKPTKAD)

• Immunoprecipitation followed by mass spectrometry:

  • Confirm that the immunoprecipitated protein is indeed SLC25A47

  • This adds an additional layer of specificity validation

How is SLC25A47 related to SIRT3 signaling and what experimental approaches can investigate this connection?

SLC25A47 appears to have a functional relationship with SIRT3 signaling pathways, particularly in the context of liver metabolism and mitochondrial function. According to the search results, SLC25A47 imports NAD+ substrate, which triggers SIRT3 activity and subsequently activates the PRKAA1/AMPK-alpha signaling cascade . This pathway ultimately downregulates sterol regulatory element-binding protein (SREBP) transcriptional activities and ATP-consuming lipogenesis to restore cellular energy balance.

To investigate this connection experimentally:

• Co-immunoprecipitation studies:

  • Use anti-SLC25A47 antibodies to pull down potential protein complexes

  • Probe for SIRT3 in the immunoprecipitated material

  • Reciprocally, use SIRT3 antibodies to pull down complexes and probe for SLC25A47

• NAD+ transport assays:

  • Measure mitochondrial NAD+ levels in wild-type versus SLC25A47-deficient models

  • Assess SIRT3 activity using deacetylation assays of known SIRT3 targets (e.g., ACSS1, IDH, GDH, SOD2)

• AMPK activation analysis:

  • Measure phosphorylation status of AMPK in response to SLC25A47 manipulation

  • Assess downstream SREBP activity and lipogenic gene expression

• Therapeutic models:

  • One publication indicates that human umbilical cord-derived mesenchymal stem cells ameliorate liver fibrosis by improving mitochondrial function via Slc25a47-Sirt3 signaling pathway

What is the current understanding of SLC25A47's role in liver metabolism based on knockout studies?

Knockout studies have provided important insights into SLC25A47's physiological role, though results have been somewhat surprising given initial hypotheses about the protein's function. Key findings from SLC25A47-deficient (Slc25a47−/−) mice include:

• Minimal metabolic phenotype:

  • No significant differences in body weight, liver weight, or food intake between wild-type and Slc25a47−/− mice on either low-fat or high-fat diets

  • No differences in hepatic triglyceride and glycogen levels

  • No significant differences in the expression of key genes involved in fatty acid oxidation (Ppara, Cpt2, Acadl, Slc25a20, Tfam)

• Improved glucose tolerance:

  • Slc25a47−/− mice showed significantly improved glucose tolerance compared to wild-type mice on high-fat diet

• Sex-specific plasma lipid effects:

  • Male but not female Slc25a47−/− mice exhibited significantly lower plasma glycerol and triglyceride levels compared to wild-type mice

  • This suggests potential sex-specific metabolic roles for SLC25A47

• Metabolomic alterations:

  • Elevated plasma levels of TCA cycle intermediates and metabolites involved in amino acid metabolism in Slc25a47−/− mice, including homocitrulline, α-ketoglutaric acid, malic acid, ureidosuccinic acid, maleic acid, fumaric acid, and N-acetylaspartic acid

  • Elevated short-chain acylcarnitines in liver tissue, including adipoylcarnitine, acetylcarnitine, butyrylcarnitine, and 2-methyl-butyroylcarnitine

These findings suggest that while SLC25A47 does impact certain aspects of metabolism, it does not function as a mitochondrial uncoupling protein as initially hypothesized.

How can SLC25A47 antibodies be used to study its regulation by PPARα in different physiological contexts?

PPARα appears to be a key regulator of SLC25A47 expression according to multiple studies. Researchers can utilize SLC25A47 antibodies to investigate this regulatory relationship across various physiological contexts using the following approaches:

• PPARα agonist/antagonist studies:

  • Treat primary hepatocytes or liver slices with PPARα agonists (e.g., Wy14643, GW7647) and analyze SLC25A47 protein levels via Western blot

  • Include appropriate controls with PPARα antagonists

  • Compare responses in different species (human, mouse, rat) as these effects appear conserved

• Nutritional regulation studies:

  • Analyze SLC25A47 protein levels during different nutritional states known to activate PPARα (fasting, high-fat feeding, etc.)

  • Use immunohistochemistry to assess liver-specific expression patterns during these interventions

• ChIP-seq validation:

  • Analyze the PPARα binding sites immediately upstream of the SLC25A47 transcriptional start site

  • Use chromatin immunoprecipitation followed by qPCR to confirm PPARα binding at these sites

• Co-expression analysis:

  • Evaluate correlation between SLC25A47 and other known PPARα target genes (CPT1A, HMGCS2, PDK4, ANGPTL4, PLIN2, FABP1) across conditions

  • Perform immunohistochemistry with antibodies against both PPARα and SLC25A47 to assess co-localization

What are the technical challenges in resolving conflicting findings about SLC25A47's role as an uncoupling protein?

The conflicting findings regarding SLC25A47's proposed role as an uncoupling protein present several technical challenges that researchers must address:

• Assay sensitivity and specificity:

  • High-resolution respirometry measurements may lack sensitivity to detect subtle uncoupling effects

  • The research by Peeters et al. used an Oroboros Oxygraph 2k for respirometry but found no uncoupling effect of SLC25A47

  • Alternative approaches such as membrane potential measurements might provide complementary data

• Expression level considerations:

  • Physiological versus overexpression systems may yield different results

  • The massive overexpression achieved in transient transfection (shown in Figure 2E of the Peeters study) may not accurately reflect physiological conditions

• Tissue and cellular context:

  • Liver-specific factors may be necessary for SLC25A47 to function as an uncoupler

  • Studies in isolated mitochondria versus intact cells/tissues may yield different results

  • Different results between in vitro and in vivo systems need reconciliation

• Compensatory mechanisms:

  • Chronic knockout models may develop compensatory mechanisms that mask phenotypes

  • Acute knockdown or inducible knockout models might reveal phenotypes not seen in constitutive knockouts

• Methodological approach to measuring uncoupling:

  • Researchers should implement multiple independent methods to assess uncoupling:

    • Respirometry

    • Membrane potential measurements

    • Thermal imaging

    • ATP production assays

    • Proton leak kinetics

The current evidence from Peeters et al. using respirometry in both cell culture and intact liver tissue does not support the uncoupling protein hypothesis for SLC25A47 , despite earlier publications suggesting this function.

How do immunohistochemistry approaches for SLC25A47 differ between normal and pathological liver tissues?

Immunohistochemical detection of SLC25A47 in normal versus pathological liver tissue requires careful consideration of several technical parameters:

• Tissue preparation optimization:

  • For the Proteintech antibody (26292-1-AP), antigen retrieval is recommended with TE buffer pH 9.0, though citrate buffer pH 6.0 can also be used as an alternative

  • Positive IHC signals have been detected in both human liver cancer tissue and normal human liver tissue

• Expression pattern differences:

  • Pay particular attention to subcellular localization changes between normal and diseased states

  • As a mitochondrial protein, normal expression should show a characteristic mitochondrial pattern

  • Changes in mitochondrial morphology or distribution in diseased states may alter staining patterns

• Quantitative analysis approaches:

  • Digital image analysis to quantify expression differences

  • Consider dual staining with mitochondrial markers to normalize for mitochondrial content changes in disease states

• Controls specific for liver pathologies:

  • Include both positive controls (normal liver) and negative controls (tissues known not to express SLC25A47)

  • Since SLC25A47 is also known as "hepatocellular carcinoma down-regulated mitochondrial carrier protein," reduced expression might be expected in HCC samples

  • One study mentions that human umbilical cord-derived mesenchymal stem cells ameliorate liver fibrosis by improving mitochondrial function via the Slc25a47-Sirt3 signaling pathway, suggesting potential relevance in fibrotic liver disease

• Recommended dilutions:

  • For the Proteintech antibody: IHC 1:50-1:500

  • For the Novus Biologicals antibody: IHC 2.5 μg/ml

What insights can metabolomic analysis provide when combined with SLC25A47 immunostaining in metabolic disease research?

Combining SLC25A47 immunostaining with metabolomic analysis offers powerful insights into the role of this protein in metabolic diseases. Based on the research findings, this integrated approach can reveal:

• Correlations between SLC25A47 expression and metabolite profiles:

  • SLC25A47-deficient mice showed elevated levels of TCA cycle intermediates in plasma (α-ketoglutaric acid, malic acid, fumaric acid)

  • Elevated short-chain acylcarnitines in liver tissue suggest alterations in fatty acid metabolism

  • Immunostaining can identify cells/regions with differential SLC25A47 expression within heterogeneous tissues that may correlate with local metabolic changes

• Sex-specific metabolic roles:

  • Male but not female Slc25a47−/− mice showed reduced plasma glycerol and triglyceride levels

  • Immunostaining combined with metabolomics in male versus female tissues could help elucidate the mechanisms behind these sex-specific effects

• Pathway analysis integration:

  • SLC25A47's connection to NAD+ transport and subsequent SIRT3 activation impacts multiple metabolic pathways

  • Correlating immunostaining intensity with downstream metabolic effects could help map the functional network of SLC25A47

• Disease progression insights:

  • Sequential sampling during disease progression (e.g., progression of liver fibrosis) with parallel immunostaining and metabolomics could reveal dynamic changes

  • The finding that umbilical cord-derived mesenchymal stem cells ameliorate liver fibrosis via Slc25a47-Sirt3 signaling suggests this pathway as a potential therapeutic target

• Therapeutic response monitoring:

  • Metabolomic changes in response to treatments that affect SLC25A47 expression or function

  • Changes in both the protein level (via immunostaining) and metabolite profiles can serve as biomarkers of intervention efficacy

This integrated approach may be particularly valuable in studying metabolic liver diseases, given SLC25A47's liver-specific expression and suggested roles in lipid metabolism.