SLC7A2 Antibody

What is SLC7A2 and what are its primary functions?

SLC7A2 (Solute Carrier Family 7 Member 2), also known as CAT2, is a member of the solute carrier superfamily that functions as a cationic amino acid transporter. It specifically transports cationic amino acids such as arginine, lysine, and ornithine into the cytosol and plays a role in regulating inflammation . SLC7A2 can be expressed in two spliced variants: CAT-2A, a low-affinity transporter constitutively expressed in liver and muscle cells, and CAT-2B, a high-affinity transporter abundantly expressed in macrophages . SLC7A2 is considered the major arginine transporter in most cells and tissues and is part of the Na+-independent transport system (system y+) that mediates L-arginine uptake .

What are the molecular characteristics of SLC7A2?

SLC7A2 has the following molecular characteristics:

• Calculated molecular weight: 698 amino acids, 76 kDa

• Observed molecular weight in experimental settings: 76 kDa

• GenBank accession number: BC104905

• Gene ID (NCBI): 6542

• UniProt ID: P52569

How is SLC7A2 expression regulated in different tissues?

SLC7A2 is expressed at relatively high levels in the skeletal muscle, placenta, and ovary under normal physiological conditions . In pathological conditions, its expression can be altered. For instance, SLC7A2 expression is downregulated in several cancer types, including non-small-cell lung cancer (NSCLC), ovarian cancer, and hepatocellular carcinoma . In macrophages, CAT-2B (a high-affinity variant of SLC7A2) is abundantly expressed and important for arginine transport, which is crucial for nitric oxide production during inflammatory responses .

What are the recommended applications for SLC7A2 antibody?

Based on the available data, SLC7A2 antibody can be used in the following applications:

It is recommended that this reagent should be titrated in each testing system to obtain optimal results . For immunohistochemistry, antigen retrieval with TE buffer pH 9.0 is suggested, with the alternative option of citrate buffer pH 6.0 .

What are the best experimental conditions for detecting SLC7A2 in different sample types?

For Western Blot analysis, SLC7A2 antibody has been validated to detect the protein in mouse and rat brain tissue samples . For detecting SLC7A2 in human samples via immunohistochemistry, stomach cancer tissue has been successfully used with the recommended antigen retrieval methods .

For cell surface detection of SLC7A2 in live intact cells, the antibody has been validated in human THP-1 monocytic leukemia cells and mouse J774 macrophage cells . When working with these cell types, using approximately 5 μg of the fluorescently labeled antibody (such as FITC-conjugated) per sample provides optimal detection .

How can I validate the specificity of an SLC7A2 antibody?

To validate the specificity of an SLC7A2 antibody, consider the following methodological approaches:

• Positive controls: Use tissues or cell lines known to express SLC7A2, such as mouse/rat brain tissue, human THP-1 monocytic leukemia cells, or mouse J774 macrophage cells .

• Molecular weight verification: Confirm that the detected band corresponds to the expected molecular weight of SLC7A2 (approximately 76 kDa) .

• Knockdown/knockout validation: Use siRNA knockdown or CRISPR-Cas9 knockout of SLC7A2 in cell lines to confirm the specificity of antibody binding.

• Peptide competition assay: Pre-incubate the antibody with the immunogen peptide (such as the peptide corresponding to amino acids 151-163 of rat SLC7A2) before application to confirm specific binding .

• Isotype controls: Include an isotype control (such as rabbit IgG-FITC for a rabbit-derived SLC7A2-FITC antibody) in flow cytometry experiments to confirm specific binding .

What is the significance of SLC7A2 expression in different cancer types?

Current research indicates that SLC7A2 may function as a tumor suppressor in several cancer types:

These findings suggest that SLC7A2 could serve as a potential biomarker for prognosis in multiple cancer types.

How does SLC7A2 affect drug sensitivity in cancer cells?

Research on NSCLC has shown that SLC7A2 plays a role in modulating drug sensitivity:

• SLC7A2 silencing enhanced the insensitivity of NSCLC cells to multiple chemotherapeutic agents, including paclitaxel, cisplatin, and gemcitabine in vitro .

• Activation of AMPK was found to upregulate SLC7A2 expression, which enhanced the sensitivity of NSCLC cells to anti-tumor drugs. This effect could be attributed to E2F1's regulation .

• In contrast, in ovarian cancer, SLC7A2 knockdown had no significant effect on the sensitivity of ovarian cancer cells to cisplatin treatment, suggesting cancer-type specific roles of SLC7A2 in drug resistance .

These findings indicate that SLC7A2 may be a potential target for overcoming drug resistance in certain cancer types, particularly NSCLC.

What is the relationship between SLC7A2 expression and immune cell infiltration in the tumor microenvironment?

Analysis of NSCLC samples has revealed correlations between SLC7A2 expression and immune cell infiltration:

The levels of SLC7A2 expression were positively correlated with the numbers of infiltrated:

• Neutrophils

• Macrophages

• Dendritic cells

Additionally, SLC7A2 expression correlated with the expression of immune cell marker genes, including:

• CD86

• HLA-DPA1

• ITGAM

This suggests that SLC7A2 may influence the tumor immune microenvironment, potentially affecting immune surveillance and response to immunotherapies. Given that SLC7A2 regulates arginine transport, which is critical for immune cell function, these correlations may reflect functional relationships in the regulation of anti-tumor immunity .

What are the recommended methods for investigating SLC7A2 function in cancer cells?

To investigate SLC7A2 function in cancer cells, consider the following methodological approaches:

• Gene expression modulation:

  • siRNA-mediated knockdown for transient reduction of SLC7A2

  • CRISPR-Cas9 for knockout studies

  • Overexpression vectors for rescue experiments

• Functional assays:

  • Cell viability assays (e.g., CCK-8) to assess proliferation

  • Colony formation assays to evaluate clonogenic potential

  • Transwell assays to measure invasion and migration capabilities

  • Drug sensitivity assays using various chemotherapeutic agents

• Molecular mechanism exploration:

  • Western blot analysis of epithelial-mesenchymal transition markers (e.g., N-cadherin, vimentin)

  • AMPK activation studies to investigate regulatory mechanisms

  • Gene Set Enrichment Analysis (GSEA) to identify enriched pathways

  • Weighted Correlation Network Analysis (WGCNA) to identify co-expressed gene modules

• Amino acid transport assays:

  • Radiolabeled amino acid uptake studies to directly measure transport function

  • Intracellular arginine concentration measurements

How can I resolve contradictory findings about SLC7A2 function in different cancer models?

When facing contradictory results about SLC7A2 function across different cancer models, consider these methodological approaches:

• Context-specific analysis:

  • Examine cancer type-specific factors that might influence SLC7A2 function

  • Consider tissue-specific splicing variants (CAT-2A vs. CAT-2B)

  • Analyze the expression of other amino acid transporters that might compensate for SLC7A2

• Comprehensive experimental design:

  • Use multiple cell lines representing the same cancer type

  • Validate in vitro findings in patient-derived xenograft models

  • Compare results across 2D and 3D culture systems

  • Consider the influence of the tumor microenvironment

• Detailed molecular characterization:

  • Determine the predominant SLC7A2 splice variant in your model

  • Assess post-translational modifications that might affect function

  • Examine subcellular localization of SLC7A2 in different contexts

• Integrative data analysis:

  • Cross-reference your findings with multiple public datasets

  • Use multi-omics approaches (transcriptomics, proteomics, metabolomics)

  • Apply pathway analysis to contextualize SLC7A2 function within broader cellular networks

For example, while SLC7A2 knockdown affected drug sensitivity in NSCLC cells , it did not significantly impact cisplatin sensitivity in ovarian cancer cells , suggesting context-dependent functions that require careful consideration of the experimental system.

What are the best approaches for studying SLC7A2 in relation to immune cell function?

Given SLC7A2's role in immune cell function, particularly through arginine transport, these methodological approaches are recommended:

• Co-culture systems:

  • Establish cancer cell-immune cell co-cultures (e.g., with macrophages, neutrophils, dendritic cells)

  • Manipulate SLC7A2 expression in either cancer cells or immune cells

  • Measure immune cell activation markers and cytokine production

• In vivo immune infiltration analysis:

  • Use immunocompetent mouse models with SLC7A2 modulation

  • Perform flow cytometry to quantify infiltrating immune cell populations

  • Conduct multiplex immunohistochemistry to assess spatial distribution

• Arginine metabolism assessment:

  • Measure nitric oxide production in macrophages co-cultured with cancer cells

  • Analyze arginase activity in the tumor microenvironment

  • Assess the impact of arginine supplementation or depletion

• Single-cell analysis:

  • Apply single-cell RNA sequencing to tumor samples with varying SLC7A2 expression

  • Identify cell-specific effects on immune populations

  • Map intercellular communication networks

For these studies, comparing results between CAT-2 deficient mice and wild-type controls can provide valuable insights, as macrophages from CAT-2 deficient mice show impaired ability to transport arginine intracellularly and produce nitric oxide in response to endotoxin .

How do the different splice variants of SLC7A2 (CAT-2A and CAT-2B) affect experimental design and interpretation?

The existence of two major splice variants of SLC7A2 introduces important considerations for experimental design:

• Variant-specific detection:

  • Ensure antibodies can distinguish between CAT-2A and CAT-2B if needed

  • Design PCR primers that can differentiate between splice variants

  • Consider the predominant variant in your tissue/cell type of interest

• Functional differences:

  • CAT-2A: Low-affinity transporter (Km ~2-5 mM), constitutively expressed in liver and muscle

  • CAT-2B: High-affinity transporter (Km ~0.1-0.4 mM), inducible in macrophages and other immune cells

  • These affinity differences affect transport kinetics and response to substrate availability

• Context-dependent regulation:

  • Consider inflammatory contexts, which may induce CAT-2B expression

  • Account for tissue-specific expression patterns in data interpretation

  • Examine regulation of each variant separately in response to experimental treatments

• Knockdown/knockout strategies:

  • Target common regions for total SLC7A2 depletion

  • Design variant-specific knockdown for more precise functional analysis

  • Validate specificity of targeting approach for the intended variant

Understanding which variant predominates in your experimental system is crucial for accurate interpretation of amino acid transport data and downstream functional effects.

What are the critical quality control parameters when working with SLC7A2 antibodies?

When working with SLC7A2 antibodies, consider these critical quality control parameters:

• Antibody validation checklist:

  • Confirm target specificity via knockout/knockdown controls

  • Verify detection at the correct molecular weight (76 kDa)

  • Test cross-reactivity with CAT-2A vs. CAT-2B splice variants

  • Validate species reactivity (human, mouse, rat) as claimed

• Application-specific considerations:

  For Western blot:

  • Optimize protein extraction methods for membrane proteins

  • Use appropriate detergents to solubilize membrane-bound SLC7A2

  • Include positive control samples (brain tissue recommended)

  For IHC/ICC:

  • Test multiple antigen retrieval methods (TE buffer pH 9.0 recommended, citrate buffer pH 6.0 as alternative)

  • Validate subcellular localization at the plasma membrane

  • Use positive control tissues with known expression

  For flow cytometry:

  • Include appropriate isotype controls

  • Use live intact cells for surface epitope detection

  • Optimize antibody concentration (~5 μg per sample suggested)

• Storage and handling:

  • Store at -20°C as recommended

  • Avoid repeated freeze-thaw cycles

  • For long-term storage, consider aliquoting

  • Note stability (typically one year after shipment)

Adherence to these parameters will help ensure reliable and reproducible results when working with SLC7A2 antibodies.

How can I optimize detection of SLC7A2 in difficult-to-analyze samples?

When working with challenging samples or low expression levels of SLC7A2, consider these optimization strategies:

• Sample preparation enhancements:

  • For membrane proteins like SLC7A2, use specialized extraction buffers containing appropriate detergents

  • Consider membrane fractionation to enrich for plasma membrane proteins

  • Optimize lysis conditions to maintain protein integrity while maximizing extraction

• Signal amplification methods:

  • For IHC/ICC: Implement tyramide signal amplification (TSA) systems

  • For Western blot: Use high-sensitivity ECL substrates or fluorescent secondary antibodies

  • For flow cytometry: Consider multi-layer staining with biotin-streptavidin systems

• Epitope accessibility improvements:

  • Test multiple antigen retrieval methods beyond the recommended ones

  • Adjust fixation protocols to preserve epitope integrity

  • For native conformation detection, consider mild fixation or live-cell approaches

• Antibody combination strategies:

  • Use multiple antibodies targeting different epitopes of SLC7A2

  • Implement co-staining with known interacting partners or membrane markers

  • Validate findings with orthogonal detection methods

• For FFPE tissue samples:

  • Extend antigen retrieval times

  • Test enzymatic pre-treatment in addition to heat-induced epitope retrieval

  • Consider thickness of sections (optimally 4-5 μm)

These approaches can help overcome technical challenges in detecting SLC7A2, particularly in samples with low expression or high background interference.