SLC22A17 Antibody

What is SLC22A17 and what are its alternative names in scientific literature?

SLC22A17 (solute carrier family 22 member 17) is a gene that encodes a transmembrane protein functioning as a receptor for lipocalin-2 (LCN2). In scientific literature, this protein is alternatively known as 24p3R, NGALR, BOCT, BOIT, 24p3 receptor, and NGAL receptor . Understanding these alternative designations is critical when conducting literature searches or cross-referencing findings across different research groups. The protein has orthologs in several mammalian species including canine, porcine, monkey, mouse and rat models, making it suitable for comparative studies across species .

What is the molecular structure and cellular localization of SLC22A17?

SLC22A17 is a transmembrane protein with a molecular mass of approximately 57.7 kilodaltons . In renal tissue, particularly in rodent models, SLC22A17 is expressed in the apical membrane of distal convoluted tubules and collecting ducts . This apical localization is significant as it positions the receptor to interact with filtered proteins in the tubular lumen. Functionally, SLC22A17 serves as a high-affinity receptor involved in protein endocytosis within the distal nephron, with binding affinity approximately 1000 times higher than that of megalin for proteins such as LCN2 or metallothionein .

What is the functional relationship between SLC22A17 and its ligand LCN2?

SLC22A17 functions as a high-affinity receptor for lipocalin-2 (LCN2/24p3/NGAL), a protein involved in iron sequestration and innate antibacterial immunity . Interestingly, these proteins exhibit an inverse regulatory relationship in response to physiological stimuli. Under hyperosmotic conditions, SLC22A17 expression is upregulated while LCN2 expression is downregulated, suggesting adaptive osmotolerant survival mechanisms . Conversely, during bacterial infections, LPS-mediated TLR4 activation increases LCN2 expression while decreasing SLC22A17, indicating a shift toward antibacterial defense . This inverse relationship appears to be mediated by the transcription factor NFAT5, which is largely responsible for the up-regulation of SLC22A17 and down-regulation of LCN2 during osmotic stress .

What are the primary research applications for SLC22A17 antibodies?

SLC22A17 antibodies are utilized across multiple experimental techniques in research settings. Based on available commercial antibodies, the primary applications include:

These applications enable researchers to investigate SLC22A17 expression levels, cellular/subcellular localization, and protein-protein interactions in various experimental systems .

How should researchers select the appropriate SLC22A17 antibody for their specific experimental needs?

When selecting an SLC22A17 antibody, researchers should consider several critical factors:

• Species reactivity: Available antibodies demonstrate varied cross-reactivity with human (Hu), mouse (Ms), rat (Rt), and other species. Select antibodies validated for your experimental model .

• Application compatibility: Verify that the antibody has been validated for your specific application (WB, ELISA, ICC, IF, IHC). Some antibodies may perform well in certain applications but poorly in others .

• Epitope targeting: Consider antibodies targeting different regions of SLC22A17. For example, antibodies targeting the middle region may provide different results than those targeting N- or C-terminal regions .

• Validation data: Prioritize antibodies with published citations or detailed validation data provided by the manufacturer, including positive and negative controls .

• Antibody format: Determine whether unconjugated or conjugated formats are needed based on your detection system requirements .

What methodological considerations are important when using SLC22A17 antibodies for cellular localization studies?

For optimal results in cellular localization studies using immunofluorescence or immunocytochemistry:

• Fixation protocol optimization: SLC22A17 is a transmembrane protein, so fixation conditions must preserve membrane structure while allowing antibody access. Compare paraformaldehyde (2-4%) with methanol fixation to determine optimal conditions for epitope preservation .

• Membrane permeabilization: For detecting intracellular epitopes, carefully optimize detergent concentration (e.g., 0.1-0.5% Triton X-100 or 0.1% saponin) to maintain membrane integrity while allowing antibody access .

• Co-localization markers: Include markers for plasma membrane (Na⁺/K⁺-ATPase) and relevant subcellular compartments to accurately assess SLC22A17 localization. This is particularly important when studying translocation between intracellular pools and the plasma membrane .

• Surface labeling techniques: To specifically detect surface-expressed SLC22A17, consider using cell-impermeant biotinylation reagents or conducting experiments at 4°C to prevent internalization during the staining procedure .

How can researchers modulate SLC22A17 expression in vitro for experimental purposes?

Based on established protocols in the literature, SLC22A17 expression can be experimentally modulated through several approaches:

• Osmotic stress induction: Increasing media osmolarity to 400-500 mosmol/L by adding 50-100 mmol/L NaCl for 6-72 hours significantly upregulates SLC22A17 expression in collecting duct cell lines like mCCD(cl.1) .

• Hormonal stimulation: Treating cells with the V2 receptor agonist dDAVP (10 nM) for 24 hours in isotonic medium increases SLC22A17 mRNA levels via cAMP/CREB signaling pathways .

• TLR4 activation: Bacterial lipopolysaccharide (LPS) treatment downregulates SLC22A17 expression while upregulating its ligand LCN2, mimicking infection conditions .

• Transcription factor manipulation: RNA interference targeting NFAT5 significantly reduces hyperosmolarity-induced SLC22A17 upregulation, while CREB inhibition using the compound 666-15 (100-250 nM) blocks dDAVP-mediated SLC22A17 induction .

These approaches provide methodological frameworks for investigating SLC22A17 regulation under different physiological and pathophysiological conditions.

What cell models are most appropriate for studying SLC22A17 function in kidney research?

The selection of appropriate cell models is critical for investigating SLC22A17 function:

• mCCD(cl.1) cells: This mouse cortical collecting duct cell line expresses SLC22A17 and demonstrates physiological responses to osmotic stress and hormonal stimulation, making it suitable for investigating regulatory mechanisms .

• mIMCD-3 cells: Mouse inner medullary collecting duct cells also express SLC22A17 and respond to osmotic stress, providing a model for studying medullary functions .

• Primary renal tubular cells: While more challenging to maintain, these provide the most physiologically relevant model for studying SLC22A17 in its native context.

When selecting a model, researchers should consider the experimental questions being addressed and the specific nephron segment of interest, as SLC22A17 expression and regulation may vary along the nephron.

How can researchers distinguish between transcriptional and post-translational regulation of SLC22A17?

To determine the regulatory mechanisms controlling SLC22A17 expression:

• mRNA vs. protein analysis: Compare changes in mRNA levels (using RT-PCR/qPCR) with protein abundance (immunoblotting) following experimental treatments. Discordant changes suggest post-translational regulation .

• Transcription factor inhibition: Employ specific inhibitors like 666-15 (CREB inhibitor) or RNA interference targeting transcription factors such as NFAT5 to assess their contribution to SLC22A17 regulation .

• Promoter analysis: Utilize luciferase reporter constructs containing the SLC22A17 promoter to directly assess transcriptional activity in response to experimental treatments.

• Protein stability assessment: Employ cycloheximide chase experiments to determine SLC22A17 protein half-life under different conditions, which can reveal changes in protein degradation rates.

• Subcellular fractionation: Separate membrane and cytosolic fractions to assess changes in SLC22A17 subcellular distribution, which may indicate regulation of trafficking rather than expression levels .

What signaling pathways regulate SLC22A17 expression and how can they be experimentally manipulated?

SLC22A17 expression is regulated by multiple signaling pathways that can be experimentally manipulated:

• NFAT5 pathway: Activated by hyperosmolarity/hypertonicity; can be manipulated through:

  • siRNA-mediated knockdown of NFAT5

  • Hyperosmotic media (400-500 mosmol/L) to activate

  • Measurement of NFAT5 nuclear translocation as experimental readout

• cAMP/CREB pathway: Activated by AVP/dDAVP; can be manipulated through:

  • dDAVP treatment (10 nM) for activation

  • 666-15 (100-250 nM) for CREB inhibition

  • PKA inhibitors to block upstream signaling

• TLR4/NFκB pathway: Activated by bacterial LPS; can be manipulated through:

  • LPS treatment for activation

  • TLR4 antagonists for inhibition

  • NFκB inhibitors to block downstream signaling

Understanding these pathways enables researchers to design experiments that can dissect the molecular mechanisms controlling SLC22A17 expression under physiological and pathophysiological conditions.

How does SLC22A17 contribute to renal physiology and pathophysiology?

The emerging functional roles of SLC22A17 in renal physiology include:

• Protein reabsorption: SLC22A17 functions as a high-affinity receptor mediating endocytosis of filtered proteins like LCN2 in the distal nephron, potentially complementing megalin-mediated endocytosis in proximal segments .

• Osmotic stress adaptation: The upregulation of SLC22A17 by hyperosmolarity, similar to AQP2, suggests its involvement in adaptive responses to osmotic stress. This may contribute to cell survival during fluctuating osmotic conditions in the renal medulla .

• Innate immunity: The inverse regulation of SLC22A17 and LCN2 during TLR4 activation suggests a role in modulating antibacterial responses during urinary tract infections .

• Cell survival regulation: The relationship between SLC22A17 and LCN2 may influence cell proliferation and damage in osmotically stressed cells, with potential implications for renal pathophysiology .

Experimental approaches to investigate these functions include knockout/knockdown studies, receptor trafficking analysis, and protein-protein interaction studies focusing on the SLC22A17-LCN2 axis.

What are the most effective methods for quantifying SLC22A17 at the protein level in experimental samples?

For accurate quantification of SLC22A17 protein in experimental samples:

• Western blotting: Optimized protocols should include:

  • Appropriate sample preparation to preserve membrane proteins

  • Careful selection of loading controls (Na⁺/K⁺-ATPase for membrane fractions)

  • Densitometric analysis with standardized exposure conditions

• Surface biotinylation assays: To specifically quantify plasma membrane SLC22A17:

  • Use cell-impermeant biotinylation reagents

  • Streptavidin pulldown of biotinylated proteins

  • Immunoblotting for SLC22A17 in the biotinylated fraction

• Flow cytometry: For cell surface quantification in non-permeabilized cells:

  • Non-permeabilized conditions for surface detection

  • Appropriate isotype controls

  • Quantitative analysis of fluorescence intensity

• ELISA: For high-throughput quantification:

  • Validate commercial kits for specificity

  • Include standard curves with recombinant protein

  • Assess matrix effects from different sample types

Each method offers different advantages, and selection should be based on the specific experimental question and available resources.

What are common challenges when working with SLC22A17 antibodies and how can they be addressed?

Researchers may encounter several challenges when working with SLC22A17 antibodies:

• Non-specific binding: Address by:

  • Optimizing blocking conditions (5% BSA may be more effective than milk for membrane proteins)

  • Including appropriate negative controls (samples from SLC22A17 knockout models or siRNA-treated cells)

  • Testing multiple antibody concentrations to find optimal signal-to-noise ratio

• Poor membrane protein extraction: Improve by:

  • Using specialized membrane protein extraction buffers containing appropriate detergents

  • Avoiding excessive heating which can cause membrane protein aggregation

  • Incorporating protease inhibitors to prevent degradation

• Inconsistent immunostaining: Enhance by:

  • Optimizing fixation protocols specifically for membrane proteins

  • Using antigen retrieval methods appropriate for the specific antibody

  • Testing different detection systems (fluorescent vs. enzymatic)

• Variable Western blot results: Standardize by:

  • Using consistent sample preparation protocols

  • Running appropriate positive controls

  • Validating results with multiple antibodies targeting different epitopes

How can researchers design experiments to study the interaction between SLC22A17 and LCN2?

To investigate SLC22A17-LCN2 interactions:

• Co-immunoprecipitation studies:

  • Immunoprecipitate SLC22A17 and probe for co-precipitated LCN2, or vice versa

  • Include appropriate controls (IgG, lysates from cells lacking either protein)

  • Consider crosslinking approaches to stabilize transient interactions

• Proximity ligation assays:

  • Visualize protein-protein interactions in situ

  • Quantify interaction signals in different cellular compartments

  • Compare interaction frequency under different experimental conditions

• Binding affinity measurements:

  • Use purified recombinant proteins for direct binding assays

  • Employ surface plasmon resonance or microscale thermophoresis

  • Determine binding constants under varying conditions (pH, ion concentration)

• Functional endocytosis assays:

  • Use fluorescently labeled LCN2 to track internalization

  • Compare uptake in cells expressing wild-type vs. mutant SLC22A17

  • Employ endocytosis inhibitors to confirm mechanism

These methodological approaches provide complementary information about the physical and functional interactions between SLC22A17 and its ligand.