SLC15A4 Antibody, FITC conjugated

What is SLC15A4 and why is it important for immunological research?

SLC15A4 is a lysosome-resident, proton-coupled histidine/oligopeptide transporter containing 12 membrane-spanning regions. It is preferentially expressed in hematopoietic lineage cells, particularly in immune cells including dendritic cells, B cells, and mast cells . SLC15A4 plays critical roles in multiple immune functions:

• Regulation of mast cell secretory-granule homeostasis and inflammatory responses

• Mediation of TLR7/8/9-triggered type I interferon (IFN-I) production

• Control of the mTORC1-TFEB signaling axis in immune cells

• Maintenance of mitochondrial integrity during cellular stress

• Recruitment of TASL (TLR Adaptor Interacting with SLC15A4 on Lysosome) to lysosomes for IRF5 activation

The protein has gained significant research interest due to its genetic association with inflammatory diseases like systemic lupus erythematosus (SLE) and colitis, positioning it as a promising therapeutic target .

What are the primary research applications for FITC-conjugated SLC15A4 antibodies?

FITC-conjugated SLC15A4 antibodies serve multiple research applications:

• Flow cytometry analysis: For quantitative assessment of SLC15A4 expression levels in different immune cell populations, particularly useful for comparing wild-type versus knockdown/knockout models

• Confocal microscopy: For visualization of SLC15A4 subcellular localization, especially its co-localization with other lysosomal proteins and binding partners like TASL

• Immunophenotyping: For identifying specific immune cell subsets based on SLC15A4 expression patterns

• Monitoring protein trafficking: For studying SLC15A4 trafficking to lysosomes, which depends on its N-terminal targeting motif and N-glycosylation at residue N436

How can I determine if SLC15A4 antibody is suitable for my specific cell type?

To determine suitability for your specific cell type:

• Review expression databases: First verify that your cell type expresses SLC15A4 (predominantly found in hematopoietic lineage cells)

• Validate with controls: Include proper positive controls (cells known to express SLC15A4, such as plasmacytoid dendritic cells) and negative controls (SLC15A4 knockout cells or SLC15A4-negative cell lines)

• Cross-validate with multiple techniques: Compare protein detection by flow cytometry with other methods like Western blotting or RT-PCR

• Check epitope conservation: Ensure the antibody targets an epitope that is conserved in your species of interest

• Test fixation compatibility: Some epitopes may be sensitive to certain fixation methods, so optimize your protocol accordingly

What protocol should I follow for optimal SLC15A4 detection by flow cytometry?

For optimal detection of SLC15A4 by flow cytometry:

• Cell preparation:

  • Isolate single-cell suspensions from relevant tissues (peritoneal lavage, spleen, or bone marrow)

  • Filter cells through a 70 μm strainer to remove aggregates

  • Count and adjust concentration to 1-5 × 10^6 cells per sample

• Surface staining (if performing dual surface/intracellular staining):

  • Wash cells in cold PBS with 2% FBS

  • Block Fc receptors with anti-CD16/32 antibody for 10 minutes at 4°C

  • Stain with surface markers (e.g., APC or FITC-conjugated anti-c-kit, PE or APC-conjugated anti-FcεRI for mast cells)

• Fixation and permeabilization (for intracellular SLC15A4):

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1% saponin or commercial permeabilization buffer

  • Note: Optimization may be required as some epitopes can be sensitive to fixation

• SLC15A4 antibody staining:

  • Incubate with FITC-conjugated anti-SLC15A4 antibody (optimal concentration determined by titration)

  • Include proper isotype control

  • Incubate for 30-45 minutes at 4°C in the dark

• Washing and analysis:

  • Wash twice with permeabilization buffer

  • Resuspend in appropriate buffer for flow cytometry analysis

  • Analyze using appropriate instrumentation (e.g., BD FACSVerse or FACSCalibur)

• Gating strategy:

  • Gate on live cells using viability dye

  • For mast cells, gate on FcεRI+c-kit+ population before analyzing SLC15A4 expression

How do I validate the specificity of SLC15A4 antibodies in my experimental system?

Validation of SLC15A4 antibody specificity is crucial for obtaining reliable results:

• Genetic controls:

  • Compare staining between wild-type cells and SLC15A4 knockout/knockdown cells (SLC15A4-KD)

  • If generating your own knockout is not feasible, RNAi-mediated knockdown can serve as an alternative

• Peptide competition assay:

  • Pre-incubate the antibody with excess SLC15A4-specific peptide

  • A significant reduction in signal indicates specificity

• Cross-validation with different antibody clones:

  • Compare results with antibodies targeting different epitopes of SLC15A4

  • Consistent results across different antibodies suggest specificity

• Immunoprecipitation followed by mass spectrometry:

  • Verify that the immunoprecipitated protein is indeed SLC15A4

  • This can also identify potential cross-reactive proteins

• Western blot analysis:

  • Confirm that the antibody detects a protein of the expected molecular weight (~68 kDa for human SLC15A4)

  • The observed band should disappear in knockout/knockdown samples

• Heterologous expression system:

  • Overexpress tagged SLC15A4 in a cell line with low/no endogenous expression

  • Compare antibody staining with tag detection

What are appropriate positive and negative controls for SLC15A4 antibody experiments?

Proper experimental controls are essential:

Positive Controls:

• Cell lines known to express high levels of SLC15A4 (e.g., CAL-1 plasmacytoid dendritic cell line)

• Primary cells with confirmed SLC15A4 expression (plasmacytoid dendritic cells, B cells, mast cells)

• Cells transfected with SLC15A4 expression vectors

• Tissues with high SLC15A4 expression (spleen, lymph nodes)

Negative Controls:

• SLC15A4 knockout or knockdown cells

• Cell lines with minimal SLC15A4 expression (verify by RNA-seq data)

• Isotype control antibody (matched to the SLC15A4 antibody's isotype)

• Secondary antibody-only controls (for indirect staining methods)

• Blocking peptide competition controls

Technical Controls:

• Fluorescence-minus-one (FMO) controls for multicolor flow cytometry

• Single-stained compensation controls for spectral overlap correction

How can I use SLC15A4 antibodies to investigate the mTORC1-TFEB signaling axis?

The SLC15A4-mTORC1-TFEB axis plays a critical role in lysosomal homeostasis and immune function. To investigate this pathway:

• Co-localization studies:

  • Use FITC-conjugated SLC15A4 antibody alongside antibodies against mTORC1 components (Raptor) and TFEB

  • Perform confocal microscopy to assess their spatial relationships in different cellular compartments

  • Look for co-localization in LAMP1+ compartments, as human SLC15A4 constitutively associates with Raptor and LAMTORs

• Phosphorylation analysis:

  • Monitor mTORC1 activity by measuring phosphorylation of S6K and 4E-BP1

  • Compare phosphorylation status in wild-type versus SLC15A4-deficient cells

  • SLC15A4 deficiency should show decreased mTORC1 activity

• TFEB nuclear translocation:

  • Use imaging or nuclear/cytoplasmic fractionation to quantify TFEB nuclear translocation

  • In SLC15A4-deficient cells, expect increased nuclear TFEB due to reduced mTORC1 activity

• CLEAR network gene expression:

  • Measure expression of TFEB-regulated genes involved in lysosomal biogenesis

  • Use RT-qPCR or RNA-seq to compare expression profiles between wild-type and SLC15A4-deficient cells

  • SLC15A4 deficiency typically increases expression of these genes

• Functional readouts:

  • Assess degranulation potential in mast cells as a functional consequence of altered TFEB activity

  • Measure cytokine production in response to TLR stimulation

What approaches can be used to study SLC15A4-TASL interactions in research settings?

SLC15A4-TASL interaction is critical for TLR7/8/9 signaling. To study this interaction:

• Co-immunoprecipitation assays:

  • Use anti-SLC15A4 antibodies to pull down protein complexes

  • Detect TASL in the immunoprecipitate by Western blotting

  • As validation, perform reciprocal co-IP with anti-TASL antibodies

• Confocal microscopy for co-localization:

  • Use FITC-conjugated SLC15A4 antibody and differently labeled TASL antibodies

  • Analyze co-localization patterns in different cellular compartments

  • Pay special attention to LAMP1+ compartments

• Mutagenesis studies:

  • Create specific mutations in key residues of SLC15A4 (e.g., E465K) or TASL (E4A, Y6A)

  • Assess their impact on protein-protein interaction using co-localization assays

  • SLC15A4 E465K mutation should abolish binding with TASL

• Domain mapping:

  • Generate truncated versions of TASL to identify minimal binding regions

  • The N-terminal helix (residues 1-14) of TASL is critical for interaction with SLC15A4

• Functional consequences:

  • Measure downstream signaling (IRF5 activation, IFN-I production) after disrupting the interaction

  • Compare responses between wild-type cells and those expressing interaction-deficient mutants

How do structural conformations of SLC15A4 affect antibody binding and experimental outcomes?

SLC15A4 can adopt different conformational states that may affect antibody binding:

• Conformation-dependent epitope accessibility:

  • SLC15A4 can exist in outward-facing (lysosomal lumen-exposed) or inward-facing (cytosol-exposed) conformations

  • Antibodies targeting luminal epitopes may show variable binding depending on the protein's conformational state

  • The conformational change occurs during TASL recruitment, as SLC15A4 switches from outward-facing to inward-facing state

• Oligomerization considerations:

  • SLC15A4 can form monomers or dimers, with the dimeric form showing an extensive interface involving cholesterol molecules

  • Dimerization may mask certain epitopes or create new conformational epitopes

  • Consider using mild detergents during sample preparation to preserve native oligomeric states

• Post-translational modifications:

  • N-glycosylation at residue N436 is important for SLC15A4 trafficking and lysosomal localization

  • Ensure your antibody's epitope is not masked by glycosylation or other modifications

  • Different fixation methods may differentially preserve these modifications

• Experiment-specific considerations:

  • For flow cytometry: Gentle fixation and permeabilization to preserve native conformations

  • For tissue sections: Consider antigen retrieval methods that may affect conformational epitopes

  • For live-cell imaging: Use antibodies against extracellular epitopes or membrane-permeable fluorescent probes

Why might I observe heterogeneous SLC15A4 staining patterns within seemingly homogeneous cell populations?

Heterogeneous staining within the same cell population may result from:

• Cell cycle-dependent expression:

  • SLC15A4 expression or localization may vary based on cell cycle stage

  • Consider cell cycle analysis in combination with SLC15A4 staining

• Activation state differences:

  • Immune cells at different activation states may have varying SLC15A4 expression levels

  • TLR stimulation can alter SLC15A4 distribution and function

• Microenvironmental influences:

  • Cells exposed to different microenvironmental cues (cytokines, nutrients) may show differential expression

  • Control for these variables in your experimental design

• Technical considerations:

  • Incomplete permeabilization leading to variable antibody access to intracellular epitopes

  • Fixation artifacts affecting epitope accessibility

  • Solution: Optimize fixation and permeabilization protocols for your specific cell type

• Biological variation in endolysosomal compartments:

  • SLC15A4 localizes to specialized endolysosomal compartments that may vary between cells

  • These compartments differ from traditional lysosomes and are characterized as lysosome-related organelles (LROs)

How can I distinguish between SLC15A4-specific effects and off-target effects in functional studies?

To distinguish specific from off-target effects:

• Genetic complementation:

  • Reintroduce wild-type SLC15A4 into knockout/knockdown cells

  • If the phenotype is rescued, it suggests the effect is SLC15A4-specific

  • Include function-deficient SLC15A4 mutants (e.g., transport-inactive mutants) as controls

• Multiple knockdown/knockout approaches:

  • Compare phenotypes obtained with different gene-silencing methods (CRISPR/Cas9, shRNA, siRNA)

  • Consistent results across methods suggest specific effects

• Structure-function analysis:

  • Use point mutations affecting specific functions of SLC15A4

  • For example, mutations affecting transport activity versus TASL binding

  • This helps delineate which function of SLC15A4 mediates the observed effect

• Dose-dependency:

  • In partial knockdown models, correlate the degree of SLC15A4 reduction with the magnitude of the phenotype

  • A clear correlation suggests specificity

• Pharmacological approach:

  • Compare genetic ablation with specific inhibitors of SLC15A4 (when available)

  • Similar phenotypes suggest target specificity of both approaches

What are potential pitfalls in analyzing SLC15A4's role in autoimmune disease models?

When studying SLC15A4 in autoimmune disease models, consider these potential pitfalls:

• Cell type-specific effects:

  • SLC15A4 functions differently across immune cell types

  • In SLE models, effects may be mediated by B cells, plasmacytoid dendritic cells, or other cell types

  • Use conditional knockout models to delineate cell type-specific contributions

• Compensatory mechanisms:

  • Long-term SLC15A4 deficiency may trigger compensatory upregulation of related transporters

  • Acute inducible knockout systems can help avoid this issue

  • Always check expression of related transporters (e.g., other SLC15 family members)

• Strain-dependent effects:

  • Genetic background can significantly influence autoimmune phenotypes

  • Always compare knockout and control mice on identical genetic backgrounds

  • Consider backcrossing to multiple strains to assess consistency

• Integration with human data:

  • Mouse phenotypes may not always translate to human disease

  • Validate findings using human samples or humanized mouse models

  • Consider that human SLC15A4 possesses pH- and temperature-dependent activity for dipeptide/tripeptide transport

• Beyond type I interferon:

  • While SLC15A4 profoundly affects IFN-I production, it also influences other pathways

  • Consider broader immune parameters, including autophagy and mitochondrial integrity

  • Assess multiple disease parameters rather than focusing solely on IFN-I-dependent readouts

How can SLC15A4 antibodies be used to investigate mitochondrial integrity in immune cells?

Recent research has revealed SLC15A4's role in maintaining mitochondrial integrity, particularly under stress conditions:

• Dual staining approaches:

  • Combine FITC-conjugated SLC15A4 antibody with mitochondrial markers

  • Use flow cytometry or imaging to correlate SLC15A4 expression with mitochondrial membrane potential

  • SLC15A4-deficient cells show decreased mitochondrial membrane potential under starvation conditions

• Stress response studies:

  • Monitor mitochondrial parameters in wild-type versus SLC15A4-deficient cells under various stressors

  • Measure oxygen consumption rate, ATP production, and ROS generation

  • Correlate findings with immune cell function and survival

• Autophagy assessment:

  • SLC15A4 is critical for autophagy sustainability but not induction

  • Use LC3 conversion assays alongside SLC15A4 staining to correlate expression with autophagic flux

  • Monitor clearance of damaged mitochondria (mitophagy) in SLC15A4-proficient versus deficient cells

• Nutrient stress experiments:

  • Examine how SLC15A4 expression affects cellular responses to amino acid starvation

  • This connects SLC15A4's transporter function with its role in maintaining cellular homeostasis under stress

• Mechanistic studies:

  • Investigate potential direct interactions between SLC15A4 and mitochondrial proteins

  • Explore how SLC15A4-dependent nutrient sensing affects mitochondrial function

  • Examine links between mTORC1 signaling, mitochondrial function, and SLC15A4 activity

What novel approaches can be used to target SLC15A4 therapeutically in autoimmune diseases?

As SLC15A4 emerges as a promising therapeutic target for autoimmune diseases:

• Structure-guided drug design:

  • The recently solved cryo-EM structures of human SLC15A4 provide a framework for rational drug design

  • Focus on compounds that:

    • Block the transport activity without affecting protein stability

    • Disrupt the SLC15A4-TASL interaction interface

    • Stabilize the outward-facing conformation to prevent TASL recruitment

• Antibody-based approaches:

  • Develop inhibitory antibodies targeting accessible epitopes of SLC15A4

  • Consider antibody-drug conjugates for cell type-specific targeting

  • Use intrabodies to target specific conformational states

• Cell type-selective delivery:

  • Design delivery systems targeting specific immune cell populations (e.g., plasmacytoid dendritic cells)

  • This could reduce off-target effects while maintaining therapeutic efficacy

  • Consider nanoparticle-based approaches for enhanced delivery

• Combination therapies:

  • Target SLC15A4 alongside other components of the TLR signaling pathway

  • Consider combining SLC15A4 inhibition with standard-of-care treatments for enhanced efficacy

  • Personalize approaches based on patient-specific SLC15A4 variants or expression patterns

• Biomarker development:

  • Use SLC15A4 antibodies to develop diagnostics identifying patients likely to respond to SLC15A4-targeted therapies

  • Monitor treatment efficacy by tracking changes in SLC15A4-dependent cellular functions

  • Develop companion diagnostics for future SLC15A4-targeted therapeutics