SLC25A21 Antibody

What is the primary biological function of SLC25A21?

SLC25A21 (Solute Carrier Family 25 Member 21) functions as a mitochondrial carrier that transports C5-C7 oxodicarboxylates across the inner mitochondrial membrane through a counter-exchange mechanism. It primarily facilitates the transport of 2-oxoadipate and 2-oxoglutarate, which are critical intermediates in lysine, tryptophan, and hydroxylysine catabolism. Within mitochondria, 2-oxoadipate is converted into acetyl-CoA, facilitating ATP production through the TCA cycle. This transport process is essential for maintaining metabolic homeostasis and energy production .

Where is SLC25A21 primarily expressed in human tissues?

SLC25A21 demonstrates ubiquitous expression across tissues but is particularly abundant in proximal tubular epithelial cells of the kidney. At the subcellular level, it is specifically localized to the inner mitochondrial membrane as a multi-pass membrane protein, consistent with its role in metabolite transport . This strategic positioning allows SLC25A21 to facilitate the exchange of metabolites between the mitochondrial matrix and the cytosol, thereby influencing cellular energy metabolism.

How is SLC25A21 gene expression regulated?

SLC25A21 expression appears to be regulated through multiple mechanisms:

• Transcriptional regulation: Evidence suggests tissue-specific transcription factors control basal expression

• Epigenetic mechanisms: DNA methylation may play a role, as reduced α-KG efflux inhibits DNA demethylases, creating a potential feedback loop affecting SLC25A21 expression

• Disease-specific dysregulation: Expression is consistently downregulated in multiple cancer types, including bladder cancer, acute myeloid leukemia, and KRAS-mutant colorectal cancer

• Metabolic state: The cellular metabolic environment appears to influence SLC25A21 expression, with palmitic acid treatment shown to decrease its expression in esophageal squamous cell carcinoma

How should researchers select appropriate SLC25A21 antibodies for specific applications?

When selecting SLC25A21 antibodies, researchers should consider:

• Target epitope: Antibodies targeting amino acids 215-264 or 72-121 of human SLC25A21 have demonstrated good specificity

• Host species and clonality: Most validated antibodies are rabbit polyclonals, which offer high sensitivity but may vary between lots

• Cross-reactivity profile: Many antibodies show cross-reactivity with multiple species, with some epitopes showing 100% identity across humans, mice, rats, and other model organisms

• Validation data: Look for antibodies with validation in multiple applications (WB, IHC, IF) and multiple cell/tissue types

• Application-specific optimization: For Western blots, antibodies typically work at 1 μg/mL; for IHC, a 1:80 dilution is often effective

What are the optimal conditions for immunohistochemical detection of SLC25A21?

For effective IHC detection of SLC25A21, follow this optimized protocol:

• Deparaffinize 3-μm tissue sections with xylene and rehydrate with descending ethanol concentrations

• Perform antigen retrieval (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

• Block endogenous peroxidase with peroxidase blocking reagent for 5 minutes

• Incubate with primary SLC25A21 antibody (1:80 dilution) at 4°C overnight

• Incubate with appropriate secondary antibody at room temperature for 1 hour

• Develop with diaminobenzidine (DAB) reagent and counterstain with hematoxylin

• Score both intensity (0-3) and proportion (0-4) of immunoreactive cells, with a total score of ≥3 considered high expression

What genetic manipulation approaches have proven effective for SLC25A21 functional studies?

Several genetic approaches have been validated for studying SLC25A21 function:

• Overexpression systems:

  • The full-length open reading frame of human SLC25A21 cloned into pcDNA3.1 vector has been successfully used

  • Stable cell lines can be established using G418 selection

  • Lentiviral vectors (LV5-SLC25A21) enable efficient transduction in hard-to-transfect cells

• Knockdown/knockout approaches:

  • siRNA targeting SLC25A21 effectively reduces expression in various cell types

  • For in vivo studies, high-pressure tail-vein injection of 70 μg Slc25a21 plasmids has been used to modulate expression

  • Multi-point in situ injection into specific tissues (e.g., renal cortex) allows for localized expression changes

• Mouse models:

  • Slc25a21(tm1a(KOMP)Wtsi) allele reduces expression by 71.3%

  • Complete knockout models (Slc25a21(tm1b) and Slc25a21(tm1d) alleles) are available but show unexpected phenotypes

How does SLC25A21 expression pattern correlate with cancer progression?

SLC25A21 expression is consistently downregulated across multiple cancer types, with significant correlations to clinical outcomes:

This consistent pattern suggests SLC25A21 functions as a tumor suppressor across multiple cancer types, making it a potential prognostic biomarker and therapeutic target .

What molecular mechanisms underlie SLC25A21's tumor-suppressive functions?

Research has revealed multiple mechanisms through which SLC25A21 exerts tumor suppression:

• Regulation of mitochondrial metabolite transport:

  • Induces efflux of mitochondrial α-KG to the cytosol

  • Disrupts glutamine-dependent anaplerosis in KRAS-mutant cancers

  • Affects metabolite influx and tricarboxylic acid cycle efficiency

• Modulation of oxidative stress and apoptosis:

  • Decreases cellular resistance against oxidative stress

  • Activates ROS-mediated mitochondrion-dependent apoptosis

  • Induces cell cycle arrest and prevents proliferation

• Genetic and signaling pathway effects:

  • Downregulates CXCL8 expression in AML

  • Impairs KRAS-mutation-mediated drug resistance

  • Influences DNA methylation patterns through α-KG-dependent demethylases

Understanding these mechanisms provides potential avenues for therapeutic intervention by restoring or mimicking SLC25A21 function in cancer cells.

How can SLC25A21 be leveraged as a biomarker in oncology research?

To effectively utilize SLC25A21 as a cancer biomarker, researchers should:

• Establish standardized detection methods:

  • Use validated antibodies with consistent dilutions (1:80 recommended for IHC)

  • Apply uniform scoring criteria combining intensity and proportion metrics

  • Employ multimodal validation (IHC, qPCR, Western blot)

• Correlate with clinical parameters:

  • Analyze association with tumor stage, grade, and invasion

  • Assess prognostic value through survival analyses

  • Consider subtype-specific expression patterns (e.g., KRAS-mutant vs. wild-type)

• Integrate with other biomarkers:

  • Combine with established markers for increased specificity

  • Consider metabolic context and related pathway markers

  • Evaluate tissue-specific expression patterns

• Therapeutic implications:

  • Monitor expression changes in response to treatment

  • Assess potential as a predictive biomarker for specific therapies

  • Consider as a target for novel therapeutic approaches

How does SLC25A21 dysfunction contribute to mitochondrial metabolic disorders?

SLC25A21 plays a critical role in mitochondrial metabolism, and its dysfunction has been implicated in several disorders:

The precise mechanisms by which SLC25A21 dysfunction leads to these conditions involve complex interplays between metabolite transport, energy production, and cellular stress responses.

What metabolomics approaches are most effective for studying SLC25A21 function?

For comprehensive metabolic analysis of SLC25A21 function, researchers should consider:

• Targeted metabolite profiling:

  • Focus on direct SLC25A21 substrates: 2-oxoadipate, 2-oxoglutarate, adipate, glutarate

  • Analyze related TCA cycle intermediates and energy metabolites (acetyl-CoA, ATP)

  • Measure lysine, tryptophan, and hydroxylysine catabolism products

• Subcellular fractionation:

  • Separate mitochondrial and cytosolic fractions to assess compartmentalization

  • Compare metabolite ratios between compartments to evaluate transport efficiency

  • Analyze effects on mitochondrial membrane potential and function

• Isotope tracing:

  • Use stable isotope-labeled precursors to track metabolic flux

  • Assess contribution to TCA cycle anaplerosis

  • Evaluate effects on energy production pathways

• Integration with functional assays:

  • Correlate metabolite changes with mitochondrial respiration measurements

  • Assess ROS production and oxidative stress markers

  • Measure cell death parameters in relation to metabolic alterations

How can researchers address discrepancies between SLC25A21 protein and mRNA expression?

When facing discrepancies between protein and transcript levels, consider these approaches:

• Technical validation:

  • Verify antibody specificity using multiple antibodies targeting different epitopes

  • Confirm primer specificity and amplification efficiency for qPCR

  • Include appropriate positive and negative controls

• Biological mechanisms:

  • Investigate post-transcriptional regulation (microRNAs, RNA-binding proteins)

  • Assess protein stability and turnover rates

  • Consider alternative splicing or protein modifications

  • Evaluate potential antisense RNA effects (e.g., SLC25A21-AS1)

• Experimental design improvements:

  • Use matched samples for protein and RNA analyses

  • Include time-course studies to capture dynamic regulation

  • Apply multiple detection methods to the same samples

  • Consider single-cell approaches to address heterogeneity

What are the critical factors for successful xenograft models studying SLC25A21 function?

For effective xenograft studies of SLC25A21 function:

• Model selection and design:

  • Choose cell lines with appropriate endogenous SLC25A21 expression levels

  • Establish stable overexpression or knockdown prior to implantation

  • Consider tissue-specific factors that may influence SLC25A21 function

• Technical considerations:

  • Verify stable expression/knockdown before implantation

  • Monitor expression throughout the experiment (expression can change in vivo)

  • Consider using inducible systems to modulate expression at specific timepoints

• Analysis parameters:

  • Assess tumor growth rate, volume, and weight

  • Evaluate metastatic potential and invasion characteristics

  • Perform immunohistochemistry on tumor sections to confirm maintained expression changes

  • Analyze metabolic parameters in tumor tissues

• Limitations to consider:

  • Immunodeficient mice lack comprehensive tumor microenvironment interactions

  • Metabolic differences between human and mouse physiology

  • Potential compensatory mechanisms that emerge in vivo

How should researchers interpret conflicting results regarding SLC25A21 function across different disease models?

When confronting apparently conflicting results:

• Context-dependent factors to consider:

  • Tissue/cell type-specific functions and expression patterns

  • Disease stage and progression status

  • Genetic background differences (e.g., KRAS mutation status)

  • Metabolic state variations between models

• Methodological considerations:

  • Differences in knockdown/overexpression efficiency

  • Variability in detection methods and antibody specificity

  • Acute versus chronic modulation effects

  • In vitro versus in vivo model discrepancies

• Resolution approaches:

  • Perform parallel experiments using identical methodologies across models

  • Investigate temporal dynamics of SLC25A21 function

  • Consider compensatory mechanisms that may emerge

  • Evaluate potential interactions with tissue-specific factors

  • Integrate multi-omics approaches to capture system-wide effects