SLC10A4 Antibody

What is SLC10A4 and why is it significant in neuroscience research?

SLC10A4 (solute carrier family 10 member 4) belongs to the bile acid:sodium symporter (BASS) family. It is primarily expressed in the brain and small intestine, with moderate expression in colon, heart, prostate, and testis . Research has established SLC10A4 as a vesicular monoaminergic and cholinergic-associated transporter critical for dopamine homeostasis and neuromodulation . The protein is localized to synaptic vesicles in cholinergic and monoaminergic neurons of both the central and peripheral nervous systems, making it a significant target for researchers investigating neural signaling mechanisms .

What is the molecular characterization of human SLC10A4?

The human SLC10A4 protein has the following characteristics:

• Full name: Solute carrier family 10 (sodium/bile acid cotransporter family), member 4

• Calculated molecular weight: 46.5 kDa (though observed at ~40 kDa in Western blots)

• Protein length: 437 amino acid residues

• Gene ID (NCBI): 201780

• GenBank Accession Number: BC019066

• UniProt ID: Q96EP9

• Chromosome location: 4p11

SLC10A4 is a multi-pass membrane protein initially characterized as a protease-activated transporter involved in bile acid uptake, but subsequent research has revealed its important role in neurotransmission .

What are the validated applications for SLC10A4 antibodies?

Based on current research, SLC10A4 antibodies have been validated for the following experimental applications:

The selection of application should be based on the specific research question and experimental design requirements .

How should researchers optimize Western blot protocols for SLC10A4 detection?

For optimal detection of SLC10A4 by Western blot:

• Sample preparation: Brain tissue (particularly mouse and rat brain) is recommended as positive control material

• Protein loading: Typically 20-50 μg total protein per lane

• Recommended dilution: Start with 1:500-1:2000 and optimize

• Expected band size: ~40 kDa (observed) vs. 47 kDa (calculated)

• Detection system: Both chemiluminescence and fluorescence-based detection systems are compatible

• Controls: Use brain tissue from SLC10A4 knockout mice as negative controls when available

Note that SLC10A4 protein consistently shows an apparent molecular weight of 30-32 kDa in mouse samples, which is below the calculated molecular weight of 47 kDa . This discrepancy is common for membrane proteins and has been reported for other members of the SLC10 carrier family as well .

How can researchers effectively investigate SLC10A4 localization in neuronal systems?

When studying SLC10A4 localization in neuronal systems, consider these methodological approaches:

• Immunofluorescence co-localization studies:

  • Co-stain with vesicular markers such as mMCP-6 for mast cells

  • Use synaptophysin as a synaptic vesicle marker

  • Employ markers for cholinergic (VAChT) or monoaminergic (VMAT2) systems

• Subcellular fractionation:

  • SLC10A4 protein can be enriched together with synaptic vesicle proteins through standard vesicle purification steps

  • Use differential centrifugation to isolate synaptic vesicle fractions

• Neuronal cell culture models:

  • SH-SY5Y (human neuroblastoma) cells show endogenous expression

  • CAD (Cath.a-differentiated) mouse cells develop neuronal phenotype with long neurite-like extensions that are immunoreactive for SLC10A4

  • Primary neuronal cultures from specific brain regions

In quantitative co-localization studies, researchers have shown that 88% of SLC10A4-positive granules overlap with mMCP-6-positive granules in mast cells, indicating its association with secretory vesicles .

What experimental approaches are recommended for studying SLC10A4 function in mast cells?

For investigating SLC10A4 function in mast cells, the following experimental approaches are effective:

• Bone marrow-derived mast cell (BMMC) isolation and culture:

  • Compare wild-type and SLC10A4 knockout mice

  • Assess degranulation via β-hexosaminidase release assays

• IgE-mediated activation models:

  • Sensitize BMMCs with anti-TNP IgE and challenge with OVA-TNP

  • Use Ca²⁺-ionophore (A23187) as positive control

• Quantification methods:

  • Measure granule-associated mediator release

  • Quantify ATP in supernatants using Luciferin-Luciferase bioluminescence assay

  • Assess prostaglandin D2 and cytokine release (e.g., IL-6)

• In vivo models:

  • Passive cutaneous anaphylaxis (PCA) in SLC10A4 knockout vs. wild-type mice

  • Compound 48/80-induced mast cell degranulation and itching behavior

Research has demonstrated that SLC10A4-deficient BMMCs show significantly reduced release of granule-associated mediators in response to IgE/antigen activation, with approximately three times less ATP detected in knockout BMMC supernatants compared to wild-type .

Why might there be discrepancies in molecular weight detection of SLC10A4?

Researchers frequently observe differences between calculated and observed molecular weights for SLC10A4:

• Post-translational modifications:

  • Glycosylation has been reported for SLC10A4

  • Protein cleavage can affect apparent molecular weight

• Technical factors:

  • Different gel systems and running conditions affect migration

  • Various tissue sources show slight variations (human vs. mouse vs. rat)

• Documented observations:

  • Calculated MW: 47 kDa

  • Commonly observed MW: 40 kDa in human samples

  • Mouse SLC10A4 protein consistently shows apparent MW of 30-32 kDa

This phenomenon is not unique to SLC10A4 but has been reported for other members of the SLC10 carrier family as well . When planning experiments, researchers should anticipate these variations and include appropriate controls.

How can researchers verify SLC10A4 antibody specificity?

To ensure antibody specificity for SLC10A4:

• Essential controls:

  • Use tissues from SLC10A4 knockout mice as negative controls

  • Include blocking peptide experiments to confirm specificity

  • Test multiple antibodies targeting different epitopes

• Cross-validation techniques:

  • Compare immunoreactivity patterns using antibodies targeting different protein regions

  • Confirm with genetic approaches (siRNA knockdown, CRISPR-Cas9)

• Known immunoreactive tissues:

  • Brain tissue (particularly cholinergic and monoaminergic regions)

  • Mast cells

  • Neuronal cell lines (SH-SY5Y, CAD)

Researchers have validated specificity using approaches such as comparing wild-type versus knockout mice immunostaining, where SLC10A4 knockout BMMCs showed complete absence of SLC10A4 protein staining .

How does SLC10A4 contribute to vesicular neurotransmitter dynamics?

SLC10A4's role in vesicular neurotransmitter dynamics involves several sophisticated mechanisms:

• Vesicular localization and transport:

  • Co-localizes with vesicular acetylcholine transporter or vesicular monoamine transporter 2

  • Influences vesicular transport efficiency of dopamine rather than direct transport

  • Affects vesicular acidification when overexpressed

• Impact on neurotransmitter homeostasis:

  • Loss of SLC10A4 results in:

    • Reduced striatal serotonin, noradrenaline, and dopamine concentrations

    • Higher dopamine turnover ratio

    • Slower dopamine clearance rates in vivo

    • Accumulation of extracellular dopamine

• Behavioral consequences:

  • SLC10A4 knockout mice display:

    • Slight hypoactivity under normal conditions

    • Hypersensitivity to amphetamine and tranylcypromine

These findings highlight SLC10A4 as a modulator of dopaminergic signaling, representing a potential target for treating neurological and mental disorders .

What is the relationship between SLC10A4 and mast cell degranulation processes?

SLC10A4 plays a complex regulatory role in mast cell function:

• Impact on degranulation mechanisms:

  • SLC10A4 is required for optimal IgE-mediated mast cell degranulation

  • SLC10A4-deficient BMMCs show significant reduction in granule-associated mediator release

  • Normal storage of mast cell protease 6 (mMCP-6) is maintained despite lack of SLC10A4

• ATP-dependent regulation:

  • SLC10A4 affects the amount of granule-associated ATP released upon IgE/antigen-induced activation

  • Live imaging shows ATP is retained to a higher degree in granules lacking SLC10A4

  • Approximately three times more ATP is detected in wild-type BMMC supernatants compared to knockouts

• Selective effects on mediator release:

  • Granule-associated mediator release is impaired in SLC10A4 knockout cells

  • Release of prostaglandin D2 and IL-6 remains normal

  • This suggests SLC10A4 specifically regulates the exocytotic release pathway

These findings have significant implications for understanding allergic responses and potentially developing targeted therapies for allergic conditions.

What are the optimal storage and handling conditions for SLC10A4 antibodies?

For maximum stability and performance of SLC10A4 antibodies:

Following these storage recommendations ensures optimal antibody performance in experimental applications .

What cell and tissue models are most suitable for studying SLC10A4 function?

Researchers investigating SLC10A4 function should consider these validated model systems:

• Neuronal models:

  • SH-SY5Y human neuroblastoma cells (endogenous expression)

  • CAD mouse cells (differentiate to neuronal phenotype)

  • Primary cultures from specific brain regions (cortex, striatum)

  • Xenopus laevis oocytes (for expression studies)

• Mast cell models:

  • Bone marrow-derived mast cells (BMMCs)

  • P815 mouse mast cell line

  • MEG-01 megakaryoblast cells

  • KU812 basophilic leukemia cells

• Tissue samples:

  • Brain tissue (highest expression)

  • Small intestine

  • Specific regions of interest: nucleus basalis magnocellularis, dorsal raphe nucleus

• Animal models:

  • SLC10A4 knockout mice for comparative studies

  • BALB/c background recommended for passive cutaneous anaphylaxis studies

These models have been experimentally validated and provide reliable systems for investigating SLC10A4 biology .

What are the emerging techniques for studying SLC10A4 transport mechanisms?

Cutting-edge approaches for investigating SLC10A4 transport function include:

• Advanced vesicular transport assays:

  • Isolated synaptic vesicle preparations from wild-type vs. knockout mice

  • Fluorescent substrate analogs for real-time transport monitoring

  • Vesicular acidification measurements using pH-sensitive fluorescent probes

• Protease activation studies:

  • Thrombin treatment to cleave the N-terminal domain

  • Investigation of protease-activated transport for taurocholic acid and lithocholic acid

  • Structure-function relationships through targeted proteolysis

• Structure-based investigations:

  • Chimeric protein approaches combining domains of SLC10A4 with other transporters (e.g., NTCP)

  • Identification of vesicular sorting domains through domain swapping experiments

  • C-terminal domain appears critical for vesicular localization vs. plasma membrane targeting

These methodologies offer promising avenues for unraveling the complex transport mechanisms of this enigmatic carrier protein .

How might SLC10A4 research contribute to understanding neurological disorders?

SLC10A4 research has significant implications for neurological and psychiatric conditions:

• Dopamine-related disorders:

  • SLC10A4 knockout mice display altered responses to psychostimulants

  • Hypersensitivity to amphetamine and tranylcypromine suggests relevance to:

    • ADHD

    • Addiction disorders

    • Depression (monoamine-related)

• Cholinergic dysfunction:

  • Altered response to cholinergic stimuli at neuromuscular junctions

  • Potential implications for:

    • Myasthenia gravis

    • Alzheimer's disease

    • Other cognitive disorders

• Mast cell-related conditions:

  • Reduced passive cutaneous anaphylaxis in SLC10A4-deficient mice

  • Less intense itching behavior in response to mast cell degranulators

  • Potential relevance to:

    • Allergic conditions

    • Inflammatory diseases

    • Pruritic disorders