SLC38A7 Antibody

What is SLC38A7 and what is its primary function in cellular physiology?

SLC38A7, also known as SNAT7 (Sodium-coupled neutral amino acid transporter 7), functions as a symporter that selectively cotransports sodium ions and amino acids, particularly L-glutamine and L-asparagine, from the lysosome into the cytoplasm . This transporter is crucial for the efflux of lysosomal degradation products and participates in mTORC1 activation . Its transport activity requires an acidic lysosomal lumen to function properly .

SLC38A7 belongs to the SLC38 family of transporters, which encode sodium-coupled amino acid transporters. Unlike other members of this family, SLC38A7 shows high selectivity for glutamine and asparagine, making it functionally distinct within its transporter family .

SLC38A7 shows a distinct expression pattern:

• Cellular expression: Expressed in all neurons but not in astrocytes in the mouse brain

• Subcellular localization:

  • Primary location: Lysosomal membrane

  • Additional locations: Cell projections, axons of neurons, neuronal cell bodies

  • Detected as a 37-40 kDa band on western blots

SLC38A7 is unique in being the first system N transporter expressed in GABAergic neurons. Its axonal localization near the synaptic cleft suggests a role in the reuptake and recycling of glutamate .

How does SLC38A7 substrate specificity differ from other SLC38 family members?

SLC38A7 exhibits a highly selective substrate profile that distinguishes it from other SLC38 family members:

• Primary substrates: L-glutamine and L-asparagine with similar transport efficiencies

• Limited transport: Unlike other SLC38 family members, some studies indicate SLC38A7 does not transport histidine , contradicting earlier findings that suggested broader substrate specificity

• Bioenergetic properties: Transport is Na⁺-dependent but, unlike some family members, does not tolerate Li⁺ substitution

These differences are methodologically important when designing transport assays, as experimental conditions must be optimized to measure the specific transport activities of SLC38A7 versus other glutamine transporters. Research approaches measuring substrate specificity have included radiolabeled substrate uptake in artificially loaded lysosomes and transstimulation assays using diverse amino acid esters .

What is the role of SLC38A7 in cancer biology and what are the implications for targeting it therapeutically?

SLC38A7 has emerged as a significant factor in cancer biology:

These findings position SLC38A7 as a potential target for glutamine-related anticancer therapies, particularly for tumors that rely on macropinocytosis of extracellular proteins as an amino acid source .

How can researchers validate SLC38A7 antibody specificity in experimental systems?

Rigorous validation of SLC38A7 antibody specificity requires multiple complementary approaches:

• CRISPR/Cas9 gene editing:

  • Homozygous disruption of the SLC38A7 gene using CRISPR/Cas9 nickase method can confirm antibody specificity by demonstrating elimination of the target band on western blots

  • Research has shown that disruption of SLC38A7 selectively abolishes a 40-kDa band on immunoblots, confirming it corresponds to native SNAT7

• Subcellular fractionation:

  • Compare the distribution profile of the SLC38A7 signal with established markers for lysosomes, mitochondria, endoplasmic reticulum, and peroxisomes

  • Validated protocols show SNAT7 strongly enriched in lysosomal fractions (L fraction), with distribution profiles matching lysosomal markers

• Functional transport assays:

  • Compare [³H]glutamine transport in wildtype versus SLC38A7-knockout samples

  • Disruption of SLC38A7 should decrease countertransport by approximately 90%

• Lysosomal disruption test:

  • Add glycine methyl ester (10 mM) to cause selective osmotic stress and lysosomal disruption

  • This should release accumulated [³H]glutamine in wildtype but not in SLC38A7-knockout samples

These validation strategies provide complementary evidence for antibody specificity and functional relevance.

What are the optimal protocols for using SLC38A7 antibodies in immunohistochemistry applications?

For optimal immunohistochemical detection of SLC38A7, researchers should follow these evidence-based methodological guidelines:

Tissue preparation:

• For paraffin-embedded sections, use either:

  • TE buffer pH 9.0 for antigen retrieval (preferred method)

  • Citric acid buffer (0.01 M, pH 6.0) heated to 100°C for 10 minutes as an alternative

Antibody incubation protocol:

• After antigen retrieval, wash sections in PBS

• Place in a humidified chamber

• Incubate with primary antibody at recommended dilutions:

  • Commercial polyclonal antibodies: 1:50-1:500 dilution range

  • Custom-made polyclonal antibodies: 1:200 dilution has been validated

• Incubate overnight at 4°C in appropriate buffer (e.g., Tris-buffered saline with 0.25% gelatin, 0.5% Triton X-100)

Visualization methods:

• For brightfield microscopy: Use HRP-conjugated secondary antibodies and appropriate substrate development

• For fluorescence microscopy: Use fluorophore-conjugated secondary antibodies (Alexa-Fluor 488-conjugated Goat Anti-Rabbit IgG has been validated)

Recommended co-staining markers for localization studies:

• Neuronal markers: NeuN (1:400 dilution)

• Astrocyte markers: GFAP (1:400 dilution)

• Dendritic markers: MAP2 (1:500 dilution)

• Synaptic markers: Synaptophysin (1:200 dilution)

What experimental strategies can be employed to study SLC38A7 transport function in lysosomes?

Studying SLC38A7 transport function in lysosomes requires specialized methodologies:

1. Lysosomal amino acid export assay based on TFEB:

• This assay uses the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis

• TFEB detects lysosomal storage and can be used to screen candidate lysosomal transporters

• The assay can detect amino acid build-up in lysosomes and their depletion by lysosomal transporters

2. Radiolabeled substrate countertransport assay:

• Prepare membrane vesicles from cell fractions enriched in lysosomes

• Preload vesicles with unlabeled substrate (e.g., glutamine)

• Measure uptake of radiolabeled substrates ([³H]glutamine, [³H]asparagine) over time

• Compare uptake between:

  • Glutamine-loaded vs. glutamine-empty fractions

  • Wildtype vs. SLC38A7-knockout samples

3. Transstimulation assays:

• Treat cell fractions with different amino acid esters

• Measure [³H]glutamine countertransport to test substrate selectivity on the luminal side

• Only true substrates will transstimulate [³H]glutamine uptake

4. Bioenergetic properties investigation:

• Test transport dependence on:

  • ATP (removal or inhibition)

  • V-type H⁺-ATPase (using specific inhibitors)

  • Lysosomal pH gradient

  • Na⁺ dependence (substitution experiments)

5. Lysosomal disruption confirmation:

• Use glycine methyl ester (10 mM) to cause osmotic disruption of lysosomes

• Verify release of accumulated [³H]glutamine as confirmation of lysosomal localization

These methodologies provide complementary approaches to characterize the substrate specificity, transport mechanism, and physiological relevance of SLC38A7 in lysosomal function.

What controls should be included when using SLC38A7 antibodies in research?

To ensure experimental rigor and reproducibility when working with SLC38A7 antibodies, researchers should implement the following controls:

Positive controls:

• Tissues with known SLC38A7 expression:

  • Mouse brain tissue (especially neurons)

  • Human cell lines: U-251 MG (human brain glioma cells), Caco-2 cells

  • Human liver tissue

Negative controls:

• Primary antibody omission controls

• Isotype controls (matched rabbit IgG at equivalent concentrations)

• Cell types known to lack expression: astrocytes for neuronal studies

Specificity controls:

• CRISPR/Cas9-mediated knockout cells or tissues when available

• RNA interference (siRNA or shRNA) to knockdown SLC38A7 expression

• Peptide competition assays using the immunizing peptide

Application-specific controls:

• For Western blot:

  • Molecular weight markers to confirm expected 37-40 kDa band

  • Loading controls (housekeeping proteins)

• For IHC/IF:

  • Secondary antibody-only controls

  • Autofluorescence controls (especially important in brain tissue)

Functional validation:

• Transport assays that measure [³H]glutamine countertransport to confirm functional relevance of detected protein

These comprehensive controls ensure that experimental findings related to SLC38A7 are specific, reproducible, and physiologically relevant.

How might recent findings about SLC38A7 inform new therapeutic approaches?

Recent research on SLC38A7 has opened several promising therapeutic avenues:

• Cancer therapeutics targeting amino acid transport:

  • SLC38A7 inhibition could starve cancer cells that rely on macropinocytosis and lysosomal protein degradation for growth in glutamine-limited environments

  • Particularly relevant for SCC, where high SLC38A7 expression correlates with poor prognosis

  • May provide selective targeting of cancer cells while sparing normal cells

• Neurological applications:

  • Given SLC38A7's role in glutamine transport in neurons and potential involvement in glutamate recycling , modulating its activity might impact:

    • Excitatory neurotransmission

    • Glutamate-glutamine cycling between neurons and glia

    • Potential implications for disorders with altered glutamatergic signaling

• Lysosomal storage disorders:

  • Better understanding of SLC38A7's role in lysosomal amino acid export could provide insights into lysosomal storage disorders

  • May inform therapeutic approaches that enhance or supplement lysosomal amino acid export

• mTORC1 signaling modulation:

  • SLC38A7 participates in mTORC1 activation

  • Targeting this pathway could impact cellular growth, autophagy, and metabolism

  • Potential applications in conditions with dysregulated mTORC1 signaling

These emerging research directions highlight the importance of continued investigation into SLC38A7 biology and the development of specific tools to modulate its activity.