SLC35B1 Antibody

What is SLC35B1 and what is its primary function?

SLC35B1 (Solute Carrier Family 35 Member B1) is an ATP:ADP antiporter that catalyzes the exchange of ATP and ADP across the endoplasmic reticulum (ER) membrane. It imports ATP from the cytosol to the ER lumen while exporting ADP in the opposite direction . This transport activity is crucial for maintaining energy homeostasis within the ER. SLC35B1 regulates ER energy metabolism and protein biogenesis by providing ATP to the ER chaperone HSPA5 (BiP), which drives protein folding and trafficking in the ER . Despite being initially classified as a nucleotide-sugar transporter (hence its synonym UDP-galactose transporter-related protein 1), its primary physiological role appears to be ATP/ADP exchange .

SLC35B1 is part of a calcium-dependent ER to cytosol low energy response axis, where calcium efflux from ER to the cytosol triggers ATP import into the ER lumen to maintain sufficient ATP supply . The protein has been found to be structurally related to members of the drug/metabolite transporter (DMT) superfamily and is predicted to have ten transmembrane helices .

What isoforms of SLC35B1 exist and how do they differ?

SLC35B1 exists in multiple isoforms, with two main variants being well-characterized:

Both isoforms function as ATP/ADP antiporters and display similar biochemical properties when tested in experimental systems . They exhibit comparable apparent Kₘₐₓ values for ATP (32.6–34.7 μM) and ADP (32.0–37.3 μM) , suggesting that they may serve redundant functions in cells depending on tissue-specific expression patterns.

Why is SLC35B1 important in cellular research?

SLC35B1 represents a critical connection between cytosolic and ER energy metabolism, with implications for:

• Protein folding and quality control mechanisms in the secretory pathway

• ER stress responses and cellular adaptation

• Energy homeostasis across cellular compartments

• Calcium signaling interactions with metabolic pathways

Disruption of SLC35B1 function leads to decelerated cell growth and induction of stress markers like CHOP, an apoptosis inducer . This indicates its essential role in cellular physiology beyond simple ATP transport. Research has shown that SLC35B1 knockdown results in reduced ER ATP levels, affecting protein biogenesis and potentially contributing to pathological states associated with ER dysfunction .

What applications are SLC35B1 antibodies validated for?

Based on available information, SLC35B1 antibodies have been validated for several experimental applications:

Commercially available antibodies like ab236909 have been specifically tested for western blotting and immunofluorescence applications with human samples . The antibody recognizes an immunogen corresponding to a recombinant fragment within human SLC35B1 amino acids 100-150 .

How can I confirm the specificity of my SLC35B1 antibody?

Confirming antibody specificity is crucial for reliable experimental results. For SLC35B1 antibodies, consider these methodological approaches:

• Positive control validation: Use purified recombinant SLC35B1 protein or lysates from cells overexpressing SLC35B1-GFP fusion proteins .

• Negative control validation: Employ lysates from SLC35B1 knockdown cells treated with siRNA targeting either the 5' UTR or coding region of SLC35B1 .

• Molecular weight verification: Confirm detection of bands at the expected molecular weight (~50 kDa for the native protein, though anomalous mobility may be observed in SDS-PAGE) .

• Cross-reactivity testing: Verify absence of signal in non-expressing tissues or in heterologous systems lacking the human protein.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to demonstrate signal elimination.

Published research has used anti-SLC35B1 antibodies to detect both endogenous protein in enriched membrane fractions and overexpressed tagged versions in experimental systems .

How should I design experiments to study SLC35B1 localization and trafficking?

When investigating SLC35B1 localization and trafficking, consider these methodological approaches:

• Co-localization studies: Use dual immunofluorescence with established ER markers (calnexin, PDI, KDEL) to confirm ER localization . SLC35B1 has been definitively localized to the ER membrane through multiple independent techniques.

• Subcellular fractionation: Employ differential centrifugation to isolate enriched ER membrane fractions followed by immunoblotting. Pancreatic rough microsomes have been used successfully as a source of enriched SLC35B1 .

• Live-cell imaging: Express fluorescently tagged SLC35B1 constructs (GFP/RFP fusions) for dynamic localization studies, though validate that tagging doesn't disrupt function or localization.

• Protease protection assays: Determine membrane topology by assessing antibody accessibility in intact vs. permeabilized cells or membrane preparations.

• Interactome analysis: Immunoprecipitation combined with mass spectrometry has revealed that SLC35B1 interacts primarily with ER proteins involved in protein import (BiP, Calnexin, Oligosaccharyltransferase, Sec61 complex, TRAP complex) and calcium homeostasis (ITPR1 and 3, SERCA2) .

When expressing tagged constructs, moderate expression levels should be maintained to avoid artifacts from overexpression .

What are the optimal conditions for detecting endogenous SLC35B1 by western blotting?

Detecting endogenous SLC35B1 by western blotting can be challenging due to its relatively low expression levels in some cell types . Optimize detection with these methodological considerations:

• Sample preparation:

  • Use enriched membrane protein extracts rather than whole cell lysates

  • Pancreatic rough microsomes provide sufficient SLC35B1 enrichment for detection

  • Avoid excessive heating of samples to prevent membrane protein aggregation

• Protein loading:

  • Load higher amounts of protein (50-100 μg) for cell types with low expression

  • HeLa cells express relatively low levels of native SLC35B1, making detection difficult

• Gel conditions:

  • Use 10-12% polyacrylamide gels for optimal resolution

  • Consider gradient gels (4-20%) for improved separation

• Transfer parameters:

  • Employ semi-dry or wet transfer with adjusted transfer time for membrane proteins

  • Use PVDF membranes rather than nitrocellulose for better protein retention

• Detection optimization:

  • Extended antibody incubation times (overnight at 4°C)

  • Enhanced chemiluminescence with longer exposure times

  • Consider using signal enhancers specifically designed for low-abundance proteins

When troubleshooting detection issues, parallel analysis of SLC35B1-overexpressing cells provides a useful positive control to confirm antibody functionality .

How can I effectively knock down or knock out SLC35B1 to study its function?

Multiple approaches have been validated for reducing SLC35B1 expression:

• siRNA knockdown:

  • Two different targeting strategies have proven effective:

    • 5' UTR-targeting siRNA (reduces expression to ~10% of control)

    • Coding region-targeting siRNA (reduces expression to ~20% of control)

  • Optimal knockdown efficiency is observed after 96 hours of treatment

  • Validation by qRT-PCR is recommended as protein detection may be difficult

• CRISPR-Cas9 gene editing:

  • Target conserved early exons to disrupt all isoforms

  • Validate knockout by genomic PCR, transcript analysis, and if possible, protein detection

  • Be aware that complete knockout may affect cell viability based on observed growth deceleration in knockdown cells

• Dominant-negative approaches:

  • Express mutated versions that incorporate identified critical residues (K117, K120, K277) shown to be important for transport function

When designing knockdown/knockout experiments, consider potential compensatory mechanisms from related transporters and include appropriate controls to monitor ER stress induction, as SLC35B1 depletion has been shown to cause slight overexpression of the apoptosis inducer CHOP .

What methods can I use to study SLC35B1 transport activity?

Investigating the transport function of SLC35B1 requires specialized techniques for measuring nucleotide exchange across membranes:

• Heterologous expression systems:

  • E. coli cells have been successfully used to express functional SLC35B1

  • Yeast (S. cerevisiae) expression systems with C-terminal GFP fusion have also been employed

  • Verify membrane integration by western blot analysis of membrane fractions

• Radiolabeled substrate transport assays:

  • [α-32P]ATP uptake into whole cells expressing SLC35B1

  • Competition assays with unlabeled nucleotides to determine substrate specificity

  • Transport kinetics analysis to determine apparent Kₘ and Vₘₐₓ values

  • Substrate saturation experiments and Eadie-Hofstee analyses

• Reconstituted proteoliposome transport:

  • Solubilize membrane proteins in detergent (n-dodecyl-β-D-maltopyranoside has been used successfully)

  • Reconstitute purified SLC35B1 into phosphatidylcholine liposomes

  • Preload liposomes with potential counter-exchange substrates (e.g., 10 mM ADP)

  • Measure uptake of radiolabeled substrates over time

  • Determine transport directionality and substrate requirements

• In vivo ER ATP measurement:

  • Use genetically encoded ATP sensors targeted to the ER lumen

  • Live cell imaging to monitor ATP levels in control vs. SLC35B1-depleted cells

Published work has demonstrated that SLC35B1 functions as an antiporter with asymmetrical apparent affinities at both sides of the membrane, exhibiting approximately 13 times higher affinity for ATP/ADP on the internal face compared to the external side .

What is known about the structure-function relationship of SLC35B1 and how can I investigate it?

While the complete three-dimensional structure of SLC35B1 has not been solved, several approaches have provided insights into structure-function relationships:

• Topology prediction and validation:

  • SLC35B1 is predicted to have ten transmembrane helices

  • It is structurally related to members of the drug/metabolite transporter (DMT) superfamily

• Conserved motif identification:

  • Bioinformatic analysis and site-directed mutagenesis have identified critical residues:

  • K117 and K120 from transmembrane helix 4 and K277 from transmembrane helix 9 play crucial roles in transport

• Isoform comparison:

  • Both isoform 1 and isoform 2 show similar transport characteristics :

    • ATP: Kₘ = 32.6–34.7 μM, Vₘₐₓ = 871.0–904.5 pmol mg protein⁻¹ h⁻¹

    • ADP: Kₘ = 32.0–37.3 μM, Vₘₐₓ = 888.4–962.3 pmol mg protein⁻¹ h⁻¹

• Site-directed mutagenesis approaches:

  • Target conserved residues in transmembrane domains

  • Characterize mutants using transport assays to determine effects on:

    • Substrate binding

    • Transport kinetics

    • Substrate specificity

• Cross-linking and accessibility studies:

  • Use cysteine-scanning mutagenesis and membrane-impermeable reagents

  • Map accessibility of residues to determine topology and substrate pathway

To further investigate structure-function relationships, researchers can employ computational modeling based on related transporters with known structures, coupled with experimental validation of predictions through mutagenesis and functional assays.

Why might I have difficulty detecting SLC35B1 in my cell line of interest?

Several factors can contribute to challenges in detecting SLC35B1:

• Expression level variation: SLC35B1 is expressed at low levels in many cell types, particularly HeLa cells . Different tissues and cell lines will exhibit varying expression levels.

• Membrane protein extraction efficiency: Standard lysis buffers may inadequately solubilize membrane proteins. Use specialized membrane protein extraction protocols with appropriate detergents.

• Antibody sensitivity limitations: Commercial antibodies may have detection thresholds above endogenous expression levels in some cell types .

• Protein degradation during processing: Membrane proteins are susceptible to degradation. Use protease inhibitors and keep samples cold throughout processing.

• Isoform-specific detection: Ensure your antibody recognizes all relevant isoforms expressed in your cell type of interest.

For improved detection:

• Use enriched membrane preparations rather than whole cell lysates

• Consider overexpression systems as positive controls

• Employ more sensitive detection methods like ECL-Prime or fluorescent secondary antibodies

• Use qRT-PCR to confirm expression at the mRNA level before attempting protein detection

How can I distinguish between specific and non-specific signals when using SLC35B1 antibodies?

To confidently distinguish between specific and non-specific signals:

• Include proper controls:

  • Positive control: Cells overexpressing SLC35B1-GFP or other tagged versions

  • Negative control: SLC35B1 knockdown cells (siRNA treatment for 96 hours provides efficient depletion)

  • Competitive inhibition: Pre-incubation of antibody with immunizing peptide

• Molecular weight verification:

  • SLC35B1 should appear at approximately 50 kDa, though anomalous mobility has been observed

  • SLC35B1-GFP fusion proteins appear at approximately 65 kDa

  • Be aware that membrane proteins may show atypical migration patterns

• Signal specificity validation:

  • Compare signals across multiple cell types with known expression differences

  • Use multiple antibodies targeting different epitopes

  • Correlate protein detection with mRNA levels determined by qRT-PCR

• Optimized experimental conditions:

  • Titrate antibody concentrations to minimize background

  • Optimize blocking conditions to reduce non-specific binding

  • Increase washing stringency to eliminate weak interactions

When interpreting immunofluorescence data, co-localization with established ER markers provides additional confirmation of specific SLC35B1 detection .

How might SLC35B1 function relate to ER stress and the unfolded protein response?

SLC35B1's role as an ATP provider to the ER has important implications for ER stress responses:

• BiP/HSPA5 function dependency: SLC35B1 provides ATP to the ER chaperone BiP (HSPA5), which drives protein folding and trafficking . Insufficient ATP supply could impair chaperone function.

• Observed stress marker induction: SLC35B1 depletion leads to slight overexpression of CHOP, an apoptosis inducer associated with ER stress .

• Growth deceleration effects: Cells with reduced SLC35B1 expression show decreased growth rates, consistent with ER stress-mediated growth inhibition .

• Calcium-energy axis connection: SLC35B1 appears to be part of a calcium-dependent ER to cytosol low energy response system , which may interact with calcium signaling aspects of the UPR.

Research approaches to investigate this relationship include:

• Monitoring UPR markers (XBP1 splicing, ATF6 cleavage, PERK phosphorylation) in SLC35B1-depleted cells

• Assessing ER protein folding capacity and secretion efficiency in cells with altered SLC35B1 levels

• Investigating potential synergistic effects between SLC35B1 inhibition and classical ER stress inducers

• Studying calcium dynamics in the context of SLC35B1 function and ER energy metabolism

Understanding these relationships could provide insights into diseases associated with ER dysfunction, including neurodegenerative disorders and diabetes.

What is known about the substrate specificity of SLC35B1 and how can I investigate it further?

SLC35B1 displays interesting substrate specificity patterns:

• Primary substrates: ATP and ADP are the primary physiological substrates, with similar apparent affinities (32-37 μM) .

• Substrate exclusivity: Competition assays revealed that SLC35B1 is highly specific for ATP and ADP, with no substantial competition from AMP, CTP, GTP, UTP, UDP-glucose, or UDP-galactose .

• Beyond strict exchange: Despite being initially characterized as a strict ATP/ADP exchanger, more recent evidence indicates SLC35B1 can function as a broader nucleotide antiporter . Additional research showed other di- and tri-nucleotides could serve as counter-substrates for ATP .

• Asymmetric affinities: The apparent affinities of SLC35B1 for ATP/ADP on the internal face were approximately 13 times higher than those on the external side , suggesting evolutionary adaptation to physiological concentration gradients.

To further investigate substrate specificity:

• Competition transport assays:

  • Use radiolabeled ATP/ADP uptake with excess unlabeled potential substrates

  • Analyze inhibition patterns to determine relative affinities

• Direct transport measurements:

  • Test uptake of various radiolabeled nucleotides

  • Assess counter-exchange capabilities in pre-loaded proteoliposomes

• Kinetic analyses:

  • Determine transport parameters for different substrates

  • Compare apparent Kₘ and Vₘₐₓ values across substrate candidates

• Structural modeling and docking:

  • Use homology models based on related transporters

  • Perform in silico docking studies with various nucleotides

  • Validate predictions through mutagenesis of predicted binding sites

This research direction is particularly relevant given the initial classification of SLC35B1 as a nucleotide-sugar transporter and subsequent findings of its role in ATP/ADP transport.