ATXN10 Antibody

What is ATXN10 and why is it significant for research?

ATXN10 (Ataxin-10) is a 475 amino acid protein belonging to the ataxin-10 family with a calculated molecular weight of approximately 53 kDa, though it typically appears around 50 kDa on Western blots . The gene is strongly expressed in the human brain, heart, skeletal muscle, kidney, and liver, as well as widely in juvenile and adult mouse brains . ATXN10 is particularly significant in research due to its association with spinocerebellar ataxia type 10 (SCA10), a slowly progressing cerebellar syndrome caused by an ATTCT pentanucleotide expansion within intron 9 . Additionally, ATXN10 is implicated in ciliopathy syndromes such as nephronophthisis (NPHP) and Joubert syndrome (JBTS), which involve disruption of cilia function leading to nephron loss, impaired renal function, and cerebellar hypoplasia .

What cellular functions has ATXN10 been implicated in?

ATXN10 plays critical roles in several cellular processes. Research has demonstrated that ATXN10 is essential for cytokinesis through its interaction with Polo Like Kinase 1 (Plk1) . Studies in neuronal cells have shown that ATXN10 can induce neuritogenesis in neuronal precursor cells by interacting with the G-protein β2 subunit to activate the RAS–MAPK–ELK-1 signaling cascade . ATXN10 is also required for embryonic heart development, and its loss results in severe cardiac developmental abnormalities leading to gestational lethality . In adult mice, ATXN10 deletion causes pancreatic, renal, and gastrointestinal abnormalities with severe defects in glucose homeostasis . Additionally, ATXN10 appears to be involved in maintaining epithelial cell identity, as its loss can trigger epithelial-to-mesenchymal transition (EMT) in kidney tubule epithelial cells .

What are the recommended applications for ATXN10 antibodies in research?

ATXN10 antibodies have been validated for multiple applications crucial for investigating this protein. The most commonly validated applications include:

When selecting an application, researchers should consider that Western blotting appears to be the most robustly validated method across multiple antibody products . For optimal results, researchers should titrate the antibody concentration in their specific experimental system, as reactivity can be sample-dependent .

What tissue or cell types have been validated for ATXN10 antibody reactivity?

ATXN10 antibodies have been tested and validated in a range of tissues and cell types. The specific reactivity profile includes:

When designing experiments, researchers should consider these validated samples, particularly when working with novel tissue or cell types where antibody performance may need additional validation .

How should I optimize antigen retrieval for ATXN10 immunohistochemistry?

For ATXN10 immunohistochemistry, optimal antigen retrieval appears to be critical for specific detection. Based on validated protocols, researchers should primarily use TE buffer at pH 9.0 for antigen retrieval . Alternatively, citrate buffer at pH 6.0 can be used, though possibly with different detection efficiency . When optimizing:

• Begin with TE buffer pH 9.0 as the primary method

• Compare results with citrate buffer pH 6.0 if needed

• Optimize incubation time and temperature based on your specific tissue sample

• Include appropriate controls (both positive and negative)

• Validate specificity with additional techniques such as Western blotting

The selection of antigen retrieval method should be determined empirically for each tissue type and fixation method, as these factors significantly impact epitope accessibility .

What controls should I include when using ATXN10 antibodies?

Proper controls are essential for interpreting ATXN10 antibody results accurately. Researchers should implement:

• Positive controls: Use tissues or cells with confirmed ATXN10 expression such as human brain tissue, HEK-293 cells, HeLa cells, human cerebellum tissue, or human kidney tissue .

• Negative controls:

  • Primary antibody omission: Incubate samples with all reagents except the primary ATXN10 antibody

  • Isotype controls: Use matched concentration of non-specific rabbit IgG

  • Blocking peptide controls: If available, pre-incubate the ATXN10 antibody with its immunizing peptide to verify signal specificity

• Sample validation controls:

  • ATXN10 knockdown/knockout samples: If possible, compare with samples where ATXN10 expression has been reduced

  • Multiple antibody validation: Use a second ATXN10 antibody targeting a different epitope to confirm findings

These controls help discriminate between specific ATXN10 detection and background or non-specific signals, which is particularly important in complex tissues with potential for cross-reactivity .

What is the subcellular localization pattern of ATXN10 and how can it be accurately detected?

ATXN10 exhibits a complex subcellular localization pattern that varies depending on cell cycle stage and cell type. Research indicates that ATXN10 is:

• Predominantly cytoplasmic in most cells

• Localizes near the centrioles and base of the primary cilium

• Shows cell cycle-dependent localization:

  • At the Golgi during interphase (phosphorylated on Serine 12)

  • At the centrioles during prophase

  • At the midbody during telophase

For accurate detection of these localization patterns, researchers should:

• Use high-resolution immunofluorescence microscopy with appropriate co-markers

• Include markers for specific organelles (e.g., FOP for centrioles)

• Consider cell cycle synchronization to examine cell cycle-dependent localization

• Use recombinant ATXN10-EGFP fusion proteins as alternative visualization tools when antibody detection is challenging

• Apply super-resolution techniques for precise colocalization studies at ciliary structures

It's worth noting that detecting endogenous ATXN10 can be challenging, as some researchers have reported difficulties using commercial antibodies for immunofluorescence detection of the native protein .

How does ATXN10 relate to cilia formation and function?

While ATXN10 has been implicated in ciliopathy syndromes, research indicates a complex relationship between ATXN10 and cilia:

• ATXN10 localizes near the base of primary cilia, suggesting a potential role in ciliary function

• Despite this localization, experimental evidence shows that loss of ATXN10 does not affect ciliogenesis in fibroblast or epithelial cells

• Interestingly, acinar cells in ATXN10 postnatal-induced mutants exhibit ectopic cilia, possibly associated with altered cell states rather than direct ciliary regulation

• ATXN10 indirectly interacts with the ciliary transition zone protein NPHP5

For researchers investigating ATXN10's relationship to cilia, methodological considerations include:

• Use multiple cilia markers (acetylated tubulin, ARL13B) alongside ATXN10 detection

• Examine both cilia formation and functional parameters

• Consider cell-type specific effects, as ATXN10's impact on cilia may vary across tissues

• Investigate potential indirect mechanisms through interaction partners like NPHP5

• Examine effects on ciliary signaling pathways rather than just structural formation

This evidence suggests that while ATXN10 is not essential for ciliogenesis, it may regulate certain aspects of ciliary function or be involved in cell-type specific ciliary processes .

How do ATXN10 mutations contribute to SCA10 and other disorders?

ATXN10 mutations are associated with several disorders through distinct mechanisms:

• Spinocerebellar ataxia type 10 (SCA10):

  • Caused by an ATTCT pentanucleotide expansion within intron 9 of the ATXN10 gene

  • The expanded repeat does not impede transcription of ATXN10 or affect splicing

  • mRNA levels appear normal in patient-derived cells

  • The pathomechanism appears to involve RNA toxicity rather than loss of ATXN10 function, as:

    • 50% reduction in ATXN10 gene dosage did not cause SCA10 phenotype in mice

    • Individuals with disruption of one ATXN10 copy due to translocation don't show ataxia

• Ciliopathy syndromes (NPHP and JBTS):

  • A protein-coding mutation (IVS8-3T > G) was found in three Turkish siblings with nephronophthisis-like kidney defects

  • These ciliopathies are characterized by nephron loss and cerebellar hypoplasia

  • The mechanism appears distinct from SCA10, possibly involving direct ATXN10 functional disruption

• Developmental abnormalities:

  • Complete loss of ATXN10 causes embryonic lethality with cardiac defects

  • Tissue-specific deletion in endothelium and myocardium is similarly lethal

  • Adult-specific deletion causes pancreatic, renal, and gastrointestinal abnormalities

For researchers studying these disorders, important methodological considerations include:

• Distinguishing between toxic RNA gain-of-function versus protein loss-of-function mechanisms

• Using appropriate disease models (expansion mutations versus null mutations)

• Considering tissue-specific effects when designing experiments

• Investigating potential sequestration of RNA-binding proteins like hnRNP K by expansion mutations

What experimental models are available for studying ATXN10-related disorders?

Researchers investigating ATXN10-related disorders have several experimental models available:

• Mouse models:

  • Congenital knockout (Atxn10^KO^): Results in embryonic lethality with cardiac defects

  • Conditional knockout (Atxn10^flox^): Allows tissue-specific or temporally controlled deletion

  • Postnatal-induced deletion: Causes pancreatic and renal abnormalities with lethality within weeks

  • Haploinsufficient mice: 50% reduction in ATXN10 does not cause SCA10 phenotype

• Cellular models:

  • Patient-derived lymphoblasts, fibroblasts, and myoblasts from SCA10 patients

  • Human-mouse somatic cell hybrids containing the SCA10 expansion

  • IMCD cells with ATXN10-EGFP expression for localization studies

  • Primary neuronal cultures (showing cytotoxicity with ATXN10 knock-down)

• Biochemical and molecular tools:

  • AUUCU RNA repeat expression systems to study toxic RNA mechanisms

  • ATXN10-EGFP fusion constructs for localization studies

  • ATXN10 antibodies for protein detection in various applications

When selecting experimental models, researchers should consider:

• Whether they are investigating RNA toxicity (SCA10) or protein function (developmental/ciliopathy)

• The relevant cellular context (neurons, kidney cells, etc.)

• The need for temporal control over ATXN10 disruption

• Species differences in ATXN10 function and expression

How can I address discrepancies between observed and calculated molecular weights for ATXN10?

Researchers often observe differences between the calculated molecular weight of ATXN10 (53 kDa) and its apparent size on Western blots (approximately 50 kDa) . This discrepancy can cause confusion when validating antibody specificity. To address this issue:

• Verify antibody specificity using:

  • Known positive control samples (human brain tissue, HEK-293, HeLa cells)

  • ATXN10 knockdown or knockout samples if available

  • Alternative antibodies targeting different epitopes

• Consider factors affecting protein migration:

  • Post-translational modifications that may alter migration patterns

  • Protein folding effects on SDS-PAGE mobility

  • Different splice variants (though major isoforms should be documented)

• Optimize detection conditions:

  • Try different gel percentages to improve resolution in the 45-55 kDa range

  • Adjust running conditions or buffer systems

  • Compare reducing and non-reducing conditions if structural elements are suspected

• Document observed molecular weight in your specific system alongside citations of previously reported weights to help establish consistency in the field .

What strategies can overcome detection challenges in neuronal tissues?

Detecting ATXN10 in neuronal tissues can be challenging due to complex tissue architecture, cross-reactivity issues, and potential low expression in specific regions. Advanced strategies include:

• Optimization of tissue preparation:

  • Test multiple fixation protocols (4% PFA, Bouin's, etc.)

  • Compare fresh frozen versus fixed tissue processing

  • Optimize section thickness (typically 5-10 μm for IHC)

• Enhanced antigen retrieval:

  • Compare heat-induced epitope retrieval methods

  • Test TE buffer pH 9.0 (recommended) versus citrate buffer pH 6.0

  • Consider enzymatic retrieval alternatives for difficult samples

• Signal amplification techniques:

  • Tyramide signal amplification for immunofluorescence

  • Polymer-based detection systems for IHC

  • Consider biotin-free detection systems to reduce background

• Reduction of background and cross-reactivity:

  • Extended blocking (3-5% BSA or normal serum)

  • Use of specialized blocking reagents for neuronal tissues

  • Careful antibody titration specific to neuronal applications

  • Pre-adsorption of antibodies with brain powder from relevant species

• Multi-antibody approach:

  • Use multiple antibodies targeting different ATXN10 epitopes

  • Validate with orthogonal techniques (Western blot, RNAscope, etc.)

Researchers should document all optimization steps and include appropriate controls to ensure reproducibility in neuronal tissue studies .

How might ATXN10 research intersect with cancer biology?

The potential intersection between ATXN10 and cancer biology represents an emerging area of investigation with several intriguing connections:

• ATXN10 and hnRNP K interactions:

  • AUUCU expansions in SCA10 sequester hnRNP K

  • hnRNP K functions as both a tumor suppressor and potential oncogene in different contexts

  • hnRNP K haploinsufficiency drives acute myeloid leukemia (AML)

  • Despite theoretical links, clinical studies have not shown significant correlation between SCA10 and cancer incidence

• ATXN10's role in cell division:

  • ATXN10 interacts with Plk1 during cytokinesis

  • Aurora B phosphorylates ATXN10, promoting this interaction

  • Disruption of cytokinesis is a recognized mechanism in cancer development

  • ATXN10's potential role in maintaining genomic stability remains underexplored

• EMT and tissue transdifferentiation:

  • ATXN10 loss triggers EMT in kidney tubule epithelial cells

  • Similar transdifferentiation occurs in pancreatic acinar cells following ATXN10 loss

  • EMT is a critical process in cancer progression and metastasis

  • The mechanisms by which ATXN10 maintains epithelial identity warrant further investigation

For researchers exploring these connections, methodological considerations include:

• Using cancer cell lines with ATXN10 manipulation (overexpression/knockdown)

• Examining ATXN10 expression patterns in cancer databases

• Investigating ATXN10's role in cellular stress responses

• Exploring potential therapeutic implications of targeting ATXN10-regulated pathways

What are the emerging techniques for studying ATXN10 protein-protein interactions?

Understanding ATXN10's functional network requires advanced methods to identify and characterize its interaction partners. Emerging techniques include:

• Proximity-based labeling approaches:

  • BioID or TurboID fusion with ATXN10 to identify proximal proteins in living cells

  • APEX2-based proximity labeling for temporal resolution of interactions

  • These methods are particularly valuable for capturing transient or context-dependent interactions

• Advanced co-immunoprecipitation strategies:

  • Crosslinking immunoprecipitation to stabilize weak or transient interactions

  • Tandem affinity purification using epitope-tagged ATXN10

  • Protein complex immunoprecipitation optimized for specific cellular compartments

• Live-cell imaging of interactions:

  • FRET/FLIM analysis of ATXN10 with potential partners

  • Split fluorescent protein complementation assays

  • Single-molecule tracking of ATXN10 complexes

• Structural biology approaches:

  • Cryo-EM analysis of ATXN10-containing complexes

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • Integrative structural modeling combining multiple data types

• Computational prediction and validation:

  • Machine learning algorithms to predict ATXN10 interaction partners

  • Network analysis of ATXN10 in protein-protein interaction databases

  • Molecular dynamics simulations of ATXN10 with predicted partners

These advanced methods can help elucidate ATXN10's interactions with proteins like G-protein β2 subunit, Plk1, and potential novel partners, providing deeper insight into its diverse cellular functions .