SLC35D3 Antibody

What is SLC35D3 and what are its primary biological functions?

SLC35D3 (Solute Carrier Family 35 Member D3) functions as a probable UDP-glucose transmembrane transporter involved in UDP-glucose transport from the cytosol to the lumen of synaptic vesicles . This protein plays several critical roles in cellular physiology:

• It is involved in platelet dense granule maturation

• Functions as a molecular adapter enhancing the formation of the PI3KC3-C1/AIC/autophagy initiation complex to promote autophagy in dopaminergic neurons

• Regulates plasma membrane localization of the D1A dopamine receptor (DRD1) and dopamine signaling

• Promotes white adipose tissue browning to ameliorate metabolic dysfunction

The protein is specifically expressed in striatonigral MSNs (medium spiny neurons) that express D1R rather than striatopallidal MSNs expressing D2R . Physically, SLC35D3 interacts with D1R, with the N-terminal portion of SLC35D3 (1-241aa) interacting with the C-terminal region of D1R (217-446aa) .

What types of SLC35D3 antibodies are available for research and what species do they react with?

Several validated SLC35D3 antibodies are available for research applications:

These antibodies detect both known isoforms of SLC35D3 . The calculated molecular weight of SLC35D3 is approximately 44 kDa, though the observed molecular weight in Western blots is reported to be around 68 kDa, possibly due to post-translational modifications .

What are the validated applications for SLC35D3 antibodies and optimal protocols for each method?

SLC35D3 antibodies have been validated for multiple experimental applications:

Western Blotting (WB):

• Sample preparation: Total protein extraction from striatum or other relevant tissues

• Loading: 20-40 μg of total protein per lane is recommended

• Detection: Total D1 receptor expression levels can be compared between wild-type and experimental samples using this method

• Note: Western blotting confirms that SLC35D3 does not affect total D1R expression levels, only its cellular distribution

Immunohistochemistry (IHC-P):

• Sample preparation: Formalin-fixed, paraffin-embedded tissues

• Antigen retrieval: Heat-mediated antigen retrieval is recommended

• Working concentration: 5 μg/ml has been validated for human small intestine tissue

• Visualization: DAB or fluorescent secondary antibody systems

Immunocytochemistry/Immunofluorescence (ICC/IF):

• Cell fixation: 4% paraformaldehyde is standard

• Working concentration: 2.5 μg/ml has been validated in HeLa cells

• Co-localization studies: Can be combined with organelle markers to determine subcellular localization (ER vs. endosomes)

How can SLC35D3 antibodies be used to study dopamine receptor trafficking?

SLC35D3 antibodies can be used in conjunction with D1R antibodies to investigate dopamine receptor trafficking mechanisms:

• Co-immunoprecipitation: Use anti-SLC35D3 antibodies to precipitate protein complexes and detect D1R by Western blot, or vice versa, to confirm physical interaction between SLC35D3 and D1R

• Immunofluorescence co-localization:

  • Double-stain cells/tissues with SLC35D3 and D1R antibodies

  • Assess co-localization using confocal microscopy

  • Quantify co-localization using appropriate software

• Subcellular fractionation and Western blotting:

  • Separate membrane fractions (plasma membrane vs. endomembrane)

  • Quantify D1R distribution using Western blot

  • Compare wild-type vs. SLC35D3 mutant/knockout samples

  • Example finding: Loss of SLC35D3 leads to increased D1R retention in endomembranes (66% in mutants vs. 48.3% in wild-type)

• Immuno-electron microscopy:

  • Gold-label D1R in tissue sections

  • Quantify gold particle distribution between plasma membrane and endomembrane structures

  • This technique revealed that SLC35D3 affects D1R trafficking but not total expression levels

How can researchers investigate the role of SLC35D3 in metabolic syndrome pathophysiology?

Research into SLC35D3's role in metabolic syndrome can be approached through several experimental strategies:

• Animal models:

  • Use of ros mutant mice (harboring SLC35D3 mutation) which display obesity and metabolic syndrome

  • Generation of adipocyte-specific SLC35D3 knockout mice using the adiponectin Cre-lox system (SAKO mice)

  • Monitoring metabolic parameters: BMI, waist circumference, blood pressure, triglycerides, cholesterol, and glucose levels

• Pharmacological intervention studies:

  • Administration of D1R agonists to SLC35D3 mutant models to assess reversibility of metabolic phenotypes

  • Monitoring improvement in metabolic parameters following intervention

• Human mutation screening:

  • Screening for SLC35D3 mutations (e.g., ΔK404, insL201) in metabolic syndrome patients

  • Protein localization studies using mutant constructs

  • Documented example: Patient #1 (Male, 55 years, BMI: 26.1, waist: 109 cm, BP: 135/85 mmHg, TG: 4.23 mmol/L) had heterozygous ΔK404 mutation

• Adipose tissue analysis:

  • Examination of SLC35D3 expression in adipose tissues of obese vs. non-obese subjects

  • Investigation of white adipose tissue browning mechanisms

  • Research has shown that SLC35D3 expression is significantly lower in adipose tissues of obese mice

What experimental approaches can be used to study SLC35D3-D1R interactions at the molecular level?

The molecular interaction between SLC35D3 and D1R can be investigated using several sophisticated techniques:

• Domain mapping through co-immunoprecipitation:

  • Create truncated constructs of SLC35D3 and D1R

  • Perform co-IP experiments to determine interacting domains

  • Research has identified that the N-terminal portion of SLC35D3 (1-241aa) interacts with the C-terminal region of D1R (217-446aa)

• Subcellular localization studies:

  • Transfect cells with wild-type or mutant SLC35D3 constructs

  • Perform co-localization studies with organelle markers (ER, endosomes, lysosomes)

  • Example finding: ΔK404 mutation causes mislocalization to LAMP3-positive late endosomes/lysosomes

• Live-cell imaging of trafficking dynamics:

  • Generate fluorescently tagged SLC35D3 and D1R constructs

  • Monitor trafficking in real-time using live-cell microscopy

  • Compare trafficking kinetics between wild-type and mutant proteins

• Functional assays of dopamine signaling:

  • Measure D1R-mediated cAMP production in cells expressing wild-type vs. mutant SLC35D3

  • Assess downstream signaling cascades through phosphorylation studies

  • Reduced plasma membrane D1R in ros striatum correlates with impaired dopamine signaling

What are common technical challenges when using SLC35D3 antibodies and how can they be addressed?

Several technical issues may arise when working with SLC35D3 antibodies:

• Discrepancy between predicted and observed molecular weight:

  • Calculated molecular weight: ~44 kDa

  • Observed molecular weight: ~68 kDa

  • Solution: This discrepancy may be due to post-translational modifications; use positive controls to confirm band specificity

• Antibody storage and stability:

  • Store antibodies at recommended temperatures (-20°C for long-term)

  • Avoid repeated freeze-thaw cycles

  • Aliquot antibodies upon receipt

  • Most antibodies are stable for one year when properly stored

• Cross-reactivity considerations:

  • SLC35D3 antibodies should not cross-react with SLC35D1 or SLC35D2

  • Verify specificity using knockout/knockdown controls when possible

  • Use appropriate blocking reagents to minimize background

• Species-specific considerations:

  • Ensure the selected antibody is validated for your species of interest

  • Consider sequence homology when working with less common model organisms

How should researchers design experiments to study mutations in SLC35D3 and their functional consequences?

When investigating SLC35D3 mutations and their functional impacts, researchers should consider:

• Expression systems for mutant proteins:

  • Generate expression constructs containing identified mutations (e.g., ΔK404, insL201)

  • Use epitope tags to facilitate detection if antibody recognition might be affected by mutations

  • Consider both transient transfection and stable cell line generation

• Subcellular localization analysis:

  • Compare wild-type vs. mutant SLC35D3 localization

  • Co-stain with organelle markers (ER, Golgi, endosomes, lysosomes)

  • Example finding: ΔK404 mutation causes mislocalization to LAMP3-positive late endosomes/lysosomes compared to wild-type

• Functional assays:

  • D1R trafficking efficiency (plasma membrane vs. intracellular ratio)

  • D1R-mediated signaling (cAMP production, downstream effector activation)

  • UDP-glucose transport activity

  • Autophagy induction capacity

• CRISPR-Cas9 genome editing:

  • Generate cellular or animal models carrying specific SLC35D3 mutations

  • Compare the effects of homozygous vs. heterozygous mutations

  • Assess tissue-specific phenotypes (adipose tissue, striatum)

How is SLC35D3 implicated in adipose tissue biology and what experimental approaches can investigate this connection?

Recent research has identified SLC35D3's role in adipose tissue biology :

• Expression analysis approaches:

  • Compare SLC35D3 expression in different adipose tissue depots

  • Analyze expression changes during diet-induced obesity

  • Note: SLC35D3 expression is significantly lower in adipose tissues of obese mice

• Adipocyte-specific knockout models:

  • Generation of adipocyte-specific SLC35D3 knockout mice using the adiponectin Cre-lox system (SAKO)

  • Phenotypic characterization including adipose tissue distribution, browning capacity, and metabolic parameters

  • Comparison with global SLC35D3 knockout models to distinguish tissue-specific effects

• White adipose tissue browning studies:

  • Analyze markers of brown/beige adipocytes in SLC35D3-deficient vs. control adipose tissue

  • Measure mitochondrial content and activity

  • Assess thermogenic capacity and energy expenditure

• Mechanistic investigations:

  • Explore the link between SLC35D3's role in dopamine signaling and adipose tissue biology

  • Investigate potential interactions with adipogenesis regulators and thermogenic pathways

What novel therapeutic approaches might target SLC35D3 for metabolic syndrome treatment?

Based on current understanding of SLC35D3 function, several therapeutic approaches could be explored:

• D1R agonist therapy:

  • Administration of D1R agonists to compensate for reduced plasma membrane D1R

  • Evidence supports that metabolic syndrome phenotype is reversible by D1R agonist administration in ros mutant mice

• SLC35D3 gene therapy approaches:

  • Delivery of functional SLC35D3 to relevant tissues (striatum, adipose tissue)

  • Development of mutation-specific approaches for patients with identified SLC35D3 mutations

• Small molecule modifiers of SLC35D3 function:

  • Compounds that enhance remaining SLC35D3 activity in heterozygous mutation carriers

  • Molecules that promote D1R trafficking through alternative pathways

• Adipose tissue browning inducers:

  • Targeting the SLC35D3-mediated pathway for white adipose tissue browning

  • Combination approaches targeting both central (striatal) and peripheral (adipose) SLC35D3 functions