SLC26A9 Antibody

What is SLC26A9 and why is it significant in cystic fibrosis research?

SLC26A9 is an anion-conducting transporter belonging to the solute carrier family 26 member 9. It serves a critical function in epithelial secretion by contributing to constitutive apical chloride conductance and enhancing cAMP-regulated CFTR currents . The protein has gained significant attention in cystic fibrosis research due to its interaction with CFTR and potential role as a disease modifier. SLC26A9 plays a central role in ion and fluid secretion in epithelial tissues affected by cystic fibrosis, particularly in airways and pancreas . Research indicates that the SLC26A9 gene may influence disease severity, making it an important target for understanding CF pathophysiology and developing therapeutic approaches .

How can I validate the specificity of an SLC26A9 antibody for immunostaining experiments?

Validating SLC26A9 antibody specificity is crucial for accurate experimental results. A recommended approach involves expressing tagged SLC26A9 in a model cell system and performing co-immunostaining with your SLC26A9 antibody and an antibody against the tag. As demonstrated in published work, researchers have validated antibody specificity by transfecting 3HA-SLC26A9 into BHK cells and co-immunostaining with anti-SLC26A9 and anti-HA antibodies . This co-localization approach allows confirmation of antibody specificity before proceeding to experimental samples.

For negative controls, you should:

• Perform immunostaining in cells known to lack SLC26A9 expression

• Use isotype control antibodies matched to your SLC26A9 antibody

• Perform peptide competition assays to demonstrate specific binding

When working with tissue samples, validation can be strengthened by comparing staining patterns with in situ hybridization data for SLC26A9 mRNA expression.

What are the main challenges in detecting endogenous SLC26A9 in primary tissues?

Detecting endogenous SLC26A9 in primary tissues presents several significant challenges:

• Low expression levels: SLC26A9 is often expressed at relatively low levels in many tissues, making detection challenging without signal amplification techniques.

• Antibody specificity issues: The lack of highly specific antibodies has been a major limitation, as evidenced by recent initiatives to develop better SLC26A9 antibodies . Cross-reactivity with other SLC26 family members is common due to sequence similarity.

• Post-translational modifications: SLC26A9 undergoes complex glycosylation with both immature (band B) and mature complex-glycosylated forms (band C), which can complicate interpretation of immunoblot data .

• Sub-cellular localization: SLC26A9 can localize to tight junctions and the plasma membrane, requiring optimization of sample preparation to preserve these structures .

These challenges have prompted researchers to develop more specific antibodies, as seen in the recent grant awarded to Dr. Anita Balázs and Dr. Frauke Stanke for developing a specific antibody against SLC26A9 .

What detection methods are most effective for analyzing SLC26A9 protein expression levels?

For comprehensive analysis of SLC26A9 protein expression, researchers should employ multiple complementary approaches:

Immunoblotting (Western Blot):

• Can distinguish between immature nonglycosylated (band B) and mature complex-glycosylated (band C) forms of SLC26A9

• Requires careful sample preparation to maintain protein integrity

• Should include positive controls with overexpressed SLC26A9 and negative controls

• Semi-quantitative when combined with densitometry analysis

Immunofluorescence Microscopy:

• Provides information on subcellular localization

• Can be combined with co-localization studies using markers for cellular compartments

• Confocal microscopy is preferred for detailed localization studies

• Requires rigorous validation of antibody specificity

Cell Surface Biotinylation:

• Specifically quantifies SLC26A9 expression at the plasma membrane

• Critical for studies examining trafficking defects

• Has been successfully used to demonstrate reduced plasma membrane expression of SLC26A9 in cells expressing F508del-CFTR

qPCR for mRNA Expression:

• Useful as a complementary approach to confirm that changes in protein levels aren't due to altered transcription

• Should be performed with carefully validated primers specific for SLC26A9

When analyzing SLC26A9 expression, it's important to note that both lysosomal and proteasomal degradation pathways can affect steady-state levels, so inhibitors like MG132 (proteasome) and chloroquine (lysosome) can be used to investigate degradation mechanisms .

How can I optimize immunoprecipitation protocols for studying SLC26A9 interactions with CFTR?

Optimizing immunoprecipitation (IP) protocols for studying SLC26A9-CFTR interactions requires careful consideration of several factors:

Lysis Buffer Composition:

• Use mild detergents (0.5-1% NP-40, Triton X-100, or CHAPS) to preserve protein-protein interactions

• Include protease inhibitors to prevent degradation

• Add phosphatase inhibitors if studying phosphorylation-dependent interactions

• Consider including low concentrations of cross-linkers to stabilize transient interactions

IP Strategy:

• For studying endogenous interactions, perform reciprocal IPs (anti-SLC26A9 followed by anti-CFTR and vice versa)

• For overexpression systems, tag one or both proteins with epitope tags (HA, FLAG, etc.) for more efficient pulldown

• Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Include appropriate negative controls (IgG control, lysates from cells lacking one protein)

Specific Considerations for SLC26A9-CFTR Interaction:

• The interaction between SLC26A9 and CFTR has been demonstrated in multiple cell types, including HEK293 cells and CFBE41o- cells

• The STAS domain of SLC26A9 interacts with the R domain of CFTR, so domain-specific antibodies might disrupt this interaction

• Both wild-type CFTR and F508del-CFTR can co-immunoprecipitate with SLC26A9, indicating physical interaction despite functional differences

Verification Methods:

• Confirm pull-down efficiency by immunoblotting 5-10% of input lysate alongside IP samples

• Consider mass spectrometry to identify additional components of the complex

• Use proximity ligation assays (PLA) as an alternative approach to verify interactions in intact cells

For studying domain-specific interactions, recombinant domain proteins have been successfully used to demonstrate interaction between the STAS domain of SLC26A9 and the R domain of CFTR through biochemical methods .

What functional assays can be used to measure SLC26A9 activity in epithelial cells?

Several functional assays can effectively measure SLC26A9 activity in epithelial cells:

FLIPR Membrane Potential (FMP) Assay:

• Reports depolarization of membrane potential due to chloride efflux

• Has been successfully used to measure SLC26A9-dependent changes in membrane potential during challenge with low-Cl- solution

• Can distinguish between SLC26A9 and CFTR activity by performing measurements in the absence and presence of forskolin

• Allows for high-throughput screening applications

Electrophysiological Methods:

Halide-Sensitive Fluorescent Indicators:

• YFP-H148Q/I152L or MQAE can be used to measure chloride transport

• Provides real-time measurements of anion transport

• Can be combined with genetic manipulation of SLC26A9 expression

Surface Expression Assays:

• Cell surface biotinylation followed by immunoblotting quantifies SLC26A9 at the plasma membrane

• This approach has demonstrated reduced plasma membrane expression of SLC26A9 in cells expressing F508del-CFTR

• Can be combined with functional assays to correlate surface expression with transport activity

When designing functional experiments, it's important to include appropriate controls to distinguish SLC26A9 activity from other anion transporters, particularly when working in systems with endogenous expression of multiple transporters.

How does F508del-CFTR mutation affect SLC26A9 expression and function?

The F508del-CFTR mutation has multiple detrimental effects on SLC26A9 expression and function through several mechanisms:

Reduced Total SLC26A9 Expression:

• Cells expressing F508del-CFTR show significantly lower steady-state levels of SLC26A9 protein compared to cells expressing wild-type CFTR or completely lacking CFTR

• This reduction occurs post-transcriptionally, as SLC26A9 mRNA levels remain unchanged

• The effect is more deleterious than the complete absence of CFTR, suggesting active degradation of SLC26A9 along with F508del-CFTR

Altered Glycosylation Profile:

• The immature, nonglycosylated form (band B) of SLC26A9 is particularly diminished in F508del-CFTR-expressing cells

• Some mature, complex-glycosylated SLC26A9 (band C) is still present, indicating that partial maturation still occurs

Reduced Plasma Membrane Expression:

• Cell-surface biotinylation assays demonstrate lower SLC26A9 expression at the plasma membrane in cells with F508del-CFTR

• This reduced surface expression correlates with diminished SLC26A9-dependent chloride conductance

Enhanced Proteasomal Degradation:

• When SLC26A9 and F508del-CFTR are co-expressed at physiological levels in primary human bronchial epithelial cells, proteasome inhibition with MG132 causes a dramatic increase in SLC26A9 immunofluorescence

• This suggests enhanced proteasomal degradation of SLC26A9 in the presence of F508del-CFTR

Mechanisms of Degradation:

• Evidence suggests two populations of SLC26A9 in F508del-CFTR expressing cells:

  • One population retained with F508del-CFTR that undergoes proteasomal degradation at the ER

  • Another non-interacting population that traffics normally to the plasma membrane

These findings have important implications for therapeutic strategies aimed at increasing SLC26A9 function in patients with the F508del-CFTR mutation, suggesting that approaches that stabilize F508del-CFTR might also benefit SLC26A9 expression and function.

What is known about SLC26A9 genetic variants affecting response to CFTR modulators?

SLC26A9 genetic variants have emerged as important modifiers of treatment response to CFTR modulators, with significant clinical implications:

rs7512462 Variant Effects:

• The SLC26A9 rs7512462 C allele is associated with greater lung function improvement in response to ivacaftor (IVA) therapy

• This variant also correlates with greater SLC26A9 expression in the pancreas and reduced gastrointestinal disease severity in CF patients

• In functional studies, the C allele demonstrates greater CFTR function after correction of CFTR-Phe508del in primary human bronchial and nasal epithelia

Mechanistic Insights:

• SLC26A9 contributes to constitutive apical chloride conductance and enhances cAMP-regulated CFTR currents

• The differential response based on SLC26A9 genotype suggests that genetic variation affects the functional interaction between SLC26A9 and pharmacologically corrected CFTR

• Research indicates differential interaction between SLC26A9 and wild-type versus Phe508del-CFTR in human bronchial epithelia

Clinical Significance:

• Genetic testing for SLC26A9 variants could potentially help predict individual responses to CFTR modulator therapy

• The data suggests that enhancing SLC26A9 function might improve lung function response to CFTR modulators targeting the Phe508del variant

• This has prompted investigation into SLC26A9 as a therapeutic target to enhance CFTR modulator efficacy

Research Directions:

• Current studies are investigating whether SLC26A9 could provide alternative chloride transport in individuals with CF genotypes for which there are no approved therapies

• Research is also exploring whether enhancing SLC26A9 function could benefit other obstructive lung diseases beyond CF

These findings highlight the importance of considering SLC26A9 genetic variants in personalized treatment approaches for CF patients and support the development of therapies targeting SLC26A9.

What strategies can rescue SLC26A9 from premature degradation in CF models?

Several promising strategies have demonstrated potential to rescue SLC26A9 from premature degradation in CF models:

CFTR Corrector Compounds:

• VX-809 (lumacaftor) treatment has been shown to increase SLC26A9 immunofluorescence in F508del/F508del primary human bronchial epithelial cells, consistent with elevated expression at the plasma membrane

• This suggests that compounds developed to correct F508del-CFTR trafficking may also benefit SLC26A9 expression

Temperature Correction:

• Low-temperature incubation (27°C), a known strategy for partially rescuing F508del-CFTR trafficking, also modestly increases SLC26A9 levels in cells co-expressing F508del-CFTR

• This provides further evidence that SLC26A9 degradation is linked to F508del-CFTR processing defects

Competitive Expression with Wild-Type CFTR:

• Co-expression of wild-type CFTR can relieve the inhibition of SLC26A9 expression caused by F508del-CFTR in a dose-dependent manner

• Functional assays confirm that SLC26A9 activity increases progressively with increasing wild-type CFTR expression

• This suggests competitive binding to SLC26A9 between wild-type and mutant CFTR

Proteasome and Lysosome Inhibition:

• Treatment with the proteasome inhibitor MG132 dramatically increases SLC26A9 immunofluorescence in F508del/F508del cells

• Lysosomal inhibition with chloroquine (CQ) also increases SLC26A9 expression, indicating degradation by both pathways

• These findings suggest potential therapeutic strategies targeting protein degradation pathways

Exploiting SLC26A9 Genetic Variants:

• The C allele of SLC26A9 rs7512462 is associated with greater CFTR function after pharmacological correction of F508del-CFTR

• This genetic insight could inform personalized approaches to enhance SLC26A9 function in CF patients

These rescue strategies highlight multiple potential therapeutic approaches to enhance SLC26A9 expression and function in CF, which might complement existing CFTR modulator therapies or provide benefit in cases where CFTR cannot be effectively targeted.

What are the current challenges in developing specific antibodies against SLC26A9?

Development of specific antibodies against SLC26A9 faces several significant challenges:

Structural Complexity:

• SLC26A9 has multiple domains including transmembrane regions, N-glycosylation sites at asparagines 153 and 156, a STAS domain, and a C-terminal PDZ domain-binding motif

• This complex structure limits the availability of unique, accessible epitopes for antibody generation

Homology with Related Proteins:

• SLC26A9 belongs to the SLC26 family with significant sequence homology to other members

• Cross-reactivity with related transporters (particularly SLC26A3 and SLC26A4) is a common issue

• The STAS domain shows particular conservation across family members

Post-translational Modifications:

• SLC26A9 undergoes complex glycosylation and exists in different glycoforms (immature band B and mature band C)

• These modifications can mask epitopes or create conformational changes affecting antibody binding

• Antibodies may preferentially recognize certain glycoforms, complicating interpretation

Low Endogenous Expression:

• Natural expression levels of SLC26A9 are relatively low in many tissues

• This makes validation in endogenous systems challenging and often requires overexpression models

Validation Difficulties:

• Limited availability of knockout models or tissues completely lacking SLC26A9 for negative control validation

• Requirement for multiple validation methods to confirm specificity

These challenges have prompted dedicated efforts to develop better antibodies, such as the recent €19,000 grant awarded to Dr. Anita Balázs and Dr. Frauke Stanke for developing a specific antibody against SLC26A9 in collaboration with Eurogentec . This initiative highlights the recognized need for better tools to detect SLC26A9 at the cellular level and investigate its function in cystic fibrosis research.

How should researchers validate newly developed SLC26A9 antibodies?

A comprehensive validation strategy for newly developed SLC26A9 antibodies should include multiple complementary approaches:

Overexpression Systems:

• Epitope-Tagged Controls:

  • Express SLC26A9 with epitope tags (HA, FLAG, etc.) in cell lines

  • Perform parallel detection with commercial anti-tag antibodies and the new SLC26A9 antibody

  • This approach has been successfully used with 3HA-SLC26A9 in BHK cells

• Western Blot Characterization:

  • Confirm detection of both immature (band B) and mature (band C) glycoforms

  • Verify molecular weight shifts with enzymatic deglycosylation

  • Compare lysates from transfected and non-transfected cells

Knockdown/Knockout Validation:

• siRNA or CRISPR-based Approaches:

  • Perform siRNA knockdown or CRISPR knockout of SLC26A9

  • Demonstrate corresponding reduction/elimination of antibody signal

  • Include non-targeting controls to confirm specificity

• Heterologous Expression:

  • Express SLC26A9 in cell lines with minimal endogenous expression

  • Demonstrate antibody signal in transfected but not parental cells

Application-Specific Validation:

• Immunofluorescence:

  • Verify subcellular localization consistent with expected distribution

  • Perform co-localization with markers for plasma membrane, tight junctions, etc.

  • Include appropriate negative controls (secondary antibody only, isotype control)

• Immunoprecipitation:

  • Confirm ability to immunoprecipitate SLC26A9 from cell lysates

  • Verify by mass spectrometry or western blot with alternative antibodies

  • Demonstrate co-immunoprecipitation of known interacting partners (e.g., CFTR)

Cross-Reactivity Testing:

• Specificity for SLC26A9:

  • Test against related SLC26 family members expressed in parallel

  • Use peptide competition assays with immunizing antigen

  • Evaluate recognition of species orthologs if cross-reactivity is claimed

• Tissue Specificity:

  • Validate expression patterns in tissues known to express SLC26A9

  • Compare with published transcriptomic data for correlation

This multi-faceted validation approach ensures confidence in antibody specificity and performance across different experimental applications, which is particularly important given the current challenges in SLC26A9 antibody development .

How might targeting SLC26A9 complement existing CFTR modulator therapies?

Targeting SLC26A9 offers several promising complementary approaches to existing CFTR modulator therapies:

Alternative Chloride Transport Pathway:

• SLC26A9 provides constitutive chloride conductance independent of CFTR activation

• This could potentially benefit patients with CFTR mutations that respond poorly to available modulators

• SLC26A9 may provide chloride transport in individuals with CFTR genotypes for which there are no approved therapies

Enhanced Efficacy of CFTR Modulators:

• Genetic evidence shows the SLC26A9 rs7512462 C allele is associated with greater lung function improvement in response to CFTR modulators

• This suggests that enhancing SLC26A9 function could potentiate the effects of CFTR-targeted therapies

• Combinatorial approaches targeting both CFTR and SLC26A9 might provide synergistic benefits

Broader Range of Affected Tissues:

• SLC26A9 plays important roles in multiple CF-affected tissues including airways and pancreas

• The rs7512462 C allele is associated with greater SLC26A9 expression in the pancreas and reduced gastrointestinal disease severity

• Targeting SLC26A9 might therefore address multiple organ manifestations of CF

Potential in Non-responsive Cases:

• In patients with minimal residual CFTR function or non-responsive mutations, SLC26A9 activation might provide alternative anion transport

• This could be particularly valuable for patients with nonsense mutations resulting in no CFTR protein product

Applications Beyond Cystic Fibrosis:

• Research is exploring whether enhancing SLC26A9 could benefit other obstructive lung diseases

• This broader therapeutic potential makes SLC26A9 an attractive complementary target

The development of specific SLC26A9 antibodies, as in the research funded by the €19,000 grant to Dr. Anita Balázs and Dr. Frauke Stanke , is a critical step toward better understanding this protein's function and developing therapeutic approaches targeting it, which could complement existing CFTR modulator therapies in CF patients.

What experimental models are most suitable for testing SLC26A9-targeting therapeutics?

Several experimental models offer distinct advantages for testing SLC26A9-targeting therapeutics:

Primary Human Airway Epithelial Cell Models:

• Primary Human Bronchial Epithelial (pHBE) Cells:

  • Gold standard for CF research with endogenous expression of both CFTR and SLC26A9

  • Allow assessment in cells from CF patients with different CFTR mutations

  • Well-differentiated cultures recapitulate in vivo epithelial structure

  • Studies have successfully examined SLC26A9 in pHBE cells from non-CF and F508del homozygote donors

  • Limited availability and variability between donors are significant drawbacks

• Primary Human Nasal Epithelial (HNE) Cells:

  • More accessible alternative to bronchial cells

  • Have been used to demonstrate greater CFTR function with the C allele of rs7512462 after correction of CFTR-Phe508del

  • Correlation with CFTR function in bronchial epithelia supports their utility

Genetically Modified Cell Lines:

• Stable Cell Lines with Variable Expression:

  • BHK cell lines stably co-expressing F508del-CFTR with varying amounts of SLC26A9 provide controlled systems for studying interaction

  • Allow isolation of clones with different expression levels for dose-response studies

  • Enable high-throughput screening approaches

• CRISPR-engineered Models:

  • Introduction of specific SLC26A9 variants (e.g., rs7512462) in isogenic backgrounds

  • Permits direct assessment of genetic variants on drug response

  • Facilitates mechanistic studies on SLC26A9-CFTR interaction

Animal Models:

• Slc26a9 Knockout Mice:

  • Slc26a9-/-/Cftr-/- double knockout models show poor post-weaning survival compared to Cftr-/- alone

  • Valuable for assessing in vivo relevance and systemic effects

  • Limited by differences between human and mouse physiology

• Transgenic Models:

  • Humanized models expressing human SLC26A9 variants

  • Can incorporate patient-specific CFTR mutations

  • Allow assessment of tissue-specific effects and systemic outcomes

Organoid Models:

• Airway Organoids:

  • 3D cultures that better recapitulate tissue architecture

  • Bridge gap between 2D cell culture and animal models

  • Enable assessment of SLC26A9 function in more physiologically relevant context

  • Allow personalized medicine approaches using patient-derived cells

The choice of model should be guided by the specific research question, with consideration of the advantages and limitations of each system. For therapeutic development, a tiered approach starting with high-throughput cell lines followed by validation in primary cells and animal models is recommended.

What are the latest advances in understanding SLC26A9 as a therapeutic target beyond cystic fibrosis?

Recent research has expanded our understanding of SLC26A9 as a therapeutic target beyond cystic fibrosis, highlighting its potential relevance in multiple conditions:

Chronic Obstructive Pulmonary Disease (COPD):

• The SLC26A9 rs7512462 CC genotype has been associated with greater lung function in individuals with COPD

• This suggests SLC26A9's chloride transport function may be protective in multiple airway diseases characterized by mucus obstruction

• Enhancing SLC26A9 function could potentially improve mucociliary clearance in COPD patients

Genetic Association Studies in General Population:

• Research has investigated SLC26A9 genetic variants and lung function in non-CF populations

• These studies suggest broader relevance of SLC26A9 in respiratory health beyond specific disease contexts

• Understanding how SLC26A9 variants affect normal physiology provides insight into potential therapeutic applications

Gastric Acid Secretion and Gastroesophageal Disorders:

• SLC26A9 plays a role in gastric acid secretion and gastric epithelial integrity

• This suggests potential applications in gastroesophageal reflux disease and related conditions

• Modulating SLC26A9 function could affect gastric pH regulation and epithelial protection

Interplay with Other Ion Channels:

• SLC26A9 associates with other ion channels beyond CFTR

• These interactions suggest potential roles in broader epithelial ion transport processes

• Targeting SLC26A9 might indirectly modulate multiple transport pathways

Emerging Molecular Understanding:

• Research has revealed that SLC26A9 is selected for endoplasmic reticulum associated degradation

• This mechanistic insight provides new approaches for stabilizing the protein

• Understanding degradation pathways enables development of strategies to enhance SLC26A9 expression

Technological Advances:

• Development of specific antibodies against SLC26A9, as in the recently funded research , will facilitate:

  • Better detection of SLC26A9 at the cellular level

  • Investigation of its function across different tissues and disease states

  • Development and validation of therapeutic compounds

  • Personalized medicine approaches based on SLC26A9 expression patterns

These advances highlight SLC26A9 as an emerging therapeutic target with potential applications extending well beyond cystic fibrosis to include multiple respiratory, gastrointestinal, and ion transport disorders.