SLC15A3 Antibody

What is SLC15A3 and what are its primary cellular functions?

SLC15A3 is a protein belonging to the solute carrier family 15 member 3, with the canonical human protein consisting of 581 amino acid residues and having a molecular mass of approximately 63.6 kDa . It functions primarily as a proton-coupled histidine and di-tripeptide transporter, predominantly localized within lysosomes of cells . SLC15A3 is a member of the Proton-dependent oligopeptide transporter (POT/PTR) protein family (TC 2.A.17) and plays significant roles in both innate immune responses and the transport of ions across cellular membranes . The protein undergoes post-translational modifications, notably glycosylation, which may impact its function and detection in experimental settings . SLC15A3 is also known by several synonyms in the literature, including PHT2, PTR3, osteoclast transporter, peptide transporter 3, peptide/histidine transporter 2, and OCTP, which researchers should be aware of when conducting literature searches .

In which tissues and cell types is SLC15A3 predominantly expressed?

SLC15A3 demonstrates a distinct expression pattern across various tissues and cell types. It is notably expressed in the urinary bladder, spleen, lung, gallbladder, and bone marrow tissues . At the cellular level, monocytes exhibit significantly higher expression of SLC15A3 compared to other immune cell populations such as T cells, NK cells, B cells, and dendritic cells . This preferential expression in monocytes is particularly important when designing experiments to study SLC15A3 function, as these cells provide the most reliable source for detecting endogenous protein . While SLC15A3 mRNA can be detected in B cells and dendritic cells after viral stimulation, the protein levels in these cells remain below detection thresholds in most experimental settings . In non-immune cell lines, SLC15A3 expression can be induced in 293T, HeLa, and HaCaT cells following viral stimulation, although baseline expression levels are typically low or undetectable . The protein is detectable in 293T cells after viral stimulation but remains undetectable in HeLa and HaCaT cells even after stimulation, highlighting the cell-specific differences in SLC15A3 protein expression .

What are the most effective applications for SLC15A3 antibodies in research?

SLC15A3 antibodies are versatile research tools that can be employed in multiple experimental applications. Western Blot (WB) represents the most common and reliable application for SLC15A3 antibodies, allowing researchers to detect both native and overexpressed protein forms while providing information about molecular weight and potential post-translational modifications . Enzyme-Linked Immunosorbent Assay (ELISA) serves as another valuable application, enabling quantitative detection of SLC15A3 in various sample types with high sensitivity . Immunohistochemistry (IHC) applications, particularly in paraffin-embedded tissues (IHC-p), allow visualization of SLC15A3 expression patterns within tissue architecture, providing spatial information about protein localization . For identifying protein-protein interactions, SLC15A3 antibodies can be effectively used in co-immunoprecipitation experiments, as demonstrated by studies showing interactions between SLC15A3 and immune signaling proteins like MAVS and STING . When selecting an antibody for specific applications, researchers should consider the validation data provided by manufacturers, including positive controls, specificity testing, and application-specific optimization protocols to ensure reliable experimental outcomes .

What factors should be considered when selecting an SLC15A3 antibody?

Selecting the appropriate SLC15A3 antibody requires careful consideration of several critical factors. First, the host species and antibody type (monoclonal vs. polyclonal) should be selected based on the experimental design; monoclonal antibodies offer high specificity for a single epitope, while polyclonal antibodies provide broader detection capability but may have higher background . Species reactivity is another crucial consideration, as SLC15A3 orthologs have been reported in mouse, rat, bovine, and chimpanzee species, and researchers must ensure the antibody recognizes the target species used in their experiments . The specific application requirements significantly influence antibody selection, as antibodies optimized for Western blotting may not perform optimally in immunohistochemistry or other applications . Researchers should carefully evaluate the detection region of the antibody (N-terminal, C-terminal, or internal domains) as this affects recognition of specific protein isoforms or fragments, particularly since SLC15A3 displays as a highly polymerized format when overexpressed . Additionally, any conjugates or tags (such as HRP, fluorescent labels, or biotinylation) should be selected based on the detection method and experimental design to optimize signal-to-noise ratios and prevent interference with protein function in functional studies .

How does SLC15A3 participate in antiviral immune responses?

SLC15A3 plays a multifaceted role in antiviral immune responses, particularly against DNA viruses like HSV-1. Overexpression studies have demonstrated that SLC15A3 significantly inhibits HSV-1 replication in host cells, as evidenced by decreased HSV-1 gD gene expression and reduced production of infectious viral particles in plaque assays . Conversely, SLC15A3 deficiency through siRNA-mediated knockdown leads to enhanced viral replication and increased production of infectious viral particles, confirming its protective function against viral infection . Mechanistically, SLC15A3 enhances type I and type III interferon responses upon viral challenge, with significantly increased transcription and protein production of IFN-β and IL29 in SLC15A3-overexpressing cells compared to controls . This enhancement of interferon production occurs even in the absence of viral stimulation, suggesting that SLC15A3 may have constitutive activity in interferon signaling pathways . Protein interaction studies have revealed that SLC15A3 physically interacts with critical components of innate immune signaling pathways, including MAVS and STING, as well as cytoplasmic DNA sensors DDX41 and cGAS, positioning it as an important regulator of multiple nucleic acid recognition pathways .

What is the role of SLC15A3 in MAVS and STING signaling pathways?

SLC15A3 serves as a positive regulator of both MAVS and STING-mediated signaling pathways, which are critical for host antiviral responses. Coimmunoprecipitation experiments have demonstrated that SLC15A3 physically interacts with both MAVS and STING adaptor proteins, suggesting direct involvement in these signaling complexes . In MAVS-mediated signaling, which primarily responds to viral RNA, co-transfection of SLC15A3 with MAVS significantly enhances IFN-β and IL29 induction following stimulation with poly(I:C), a synthetic RNA analog . Similarly, in STING-mediated signaling, which responds to cytosolic DNA, co-transfection of SLC15A3 with STING potentiates IFN-β and IL29 production upon stimulation with HSV60, a synthetic double-stranded DNA fragment . These findings indicate that SLC15A3 functions as an important amplifier of both RNA and DNA sensing pathways in innate immunity . The enhancement of both pathways by SLC15A3 suggests that it may act at a convergence point or through a common mechanism affecting both MAVS and STING-dependent signaling cascades . Understanding the exact molecular mechanism by which SLC15A3 potentiates these pathways requires further investigation, but current evidence indicates that it may facilitate signaling complex formation, enhance signal transduction efficiency, or regulate the subcellular localization of key signaling components .

What are the optimal protocols for detecting SLC15A3 in different experimental systems?

Detecting SLC15A3 presents unique challenges due to its cell type-specific expression patterns and induction characteristics. For primary human monocytes, which express the highest levels of SLC15A3, direct lysis in RIPA buffer supplemented with protease inhibitors followed by Western blotting provides reliable detection of the endogenous protein, especially after viral stimulation . In cell lines like 293T where basal expression is low, viral stimulation (such as HSV-1 at MOI of 0.01-0.1) for 6-24 hours significantly increases SLC15A3 expression, making it detectable by Western blot . For immunoprecipitation experiments, epitope-tagged SLC15A3 constructs (such as HA-tagged) transfected into 293T cells have proven effective for studying protein interactions with immune signaling components . When detecting SLC15A3 by Western blot, researchers should be aware that overexpressed SLC15A3 displays as a highly polymerized format, which may affect its migration pattern on SDS-PAGE gels . For mRNA detection, quantitative RT-PCR remains the most sensitive method for detecting SLC15A3 expression across different cell types, including those where protein levels remain below detection thresholds . In tissue samples, immunohistochemistry using paraffin-embedded sections has been successfully employed, with urinary bladder, spleen, lung, gallbladder, and bone marrow tissues showing the most robust signals .

How can researchers effectively validate the specificity of SLC15A3 antibodies?

Validating antibody specificity for SLC15A3 requires a multi-faceted approach to ensure reliable experimental results. The most definitive validation approach involves comparing antibody reactivity in SLC15A3 knockout versus wild-type samples, either using CRISPR/Cas9-mediated knockout cell lines or siRNA-mediated knockdown systems as demonstrated in previous studies . Overexpression systems serve as positive controls, where cells transfected with SLC15A3 expression vectors display significantly enhanced signal compared to empty vector controls, confirming antibody specificity . Peptide competition assays provide another layer of validation, where pre-incubation of the antibody with the immunizing peptide should abolish specific binding in subsequent detection experiments . Cross-reactivity testing against related proteins, particularly other members of the SLC15 family, helps ensure the antibody specifically recognizes SLC15A3 rather than structurally similar proteins . Cell type-specific expression patterns can also be used for validation, as antibodies should show the strongest signals in monocytes compared to other immune cells, consistent with known expression profiles . For polyclonal antibodies, batch-to-batch variation should be carefully assessed through comparative testing of different lots using the same experimental conditions and samples . Finally, orthogonal validation using different detection methods (such as mass spectrometry) can provide definitive confirmation of antibody specificity when resources permit .

What are the common challenges and troubleshooting strategies for SLC15A3 antibody experiments?

SLC15A3 antibody experiments present several unique challenges that require specific troubleshooting strategies. One major challenge is the cell type-dependent expression of SLC15A3, with protein being undetectable in many cell types even after stimulation . Researchers can address this by using monocytes for endogenous protein studies or by employing viral stimulation to upregulate expression in amenable cell types like 293T . High molecular weight bands or smears may appear in Western blots due to SLC15A3's tendency to form highly polymerized structures when overexpressed, which can be partially resolved by optimizing sample denaturation conditions or using gradient gels for better separation . Non-specific background in immunohistochemistry applications can be reduced by extending blocking steps, optimizing antibody dilutions, and including appropriate controls for tissue autofluorescence . For co-immunoprecipitation experiments studying SLC15A3 interactions with signaling proteins, weak or transient interactions may require chemical crosslinking prior to cell lysis or modification of buffer conditions to stabilize protein complexes . When studying the functional role of SLC15A3 in interferon responses, the timing of analysis is critical as both early (2-6 hours) and late (12-24 hours) time points post-stimulation provide valuable but different insights into the regulatory mechanisms . Finally, for quantitative comparisons across different experimental conditions, normalization to appropriate housekeeping proteins and inclusion of positive controls (such as cells stimulated with known interferon inducers) are essential for reliable interpretation of results .