ALYREF Antibody

What is ALYREF and why is it important in cancer research?

ALYREF is an RNA-binding protein involved in transcriptional regulation and nuclear mRNA export. It has emerged as a significant factor in cancer biology, particularly in breast carcinogenesis. Research has identified ALYREF gene amplification in human cancers, with high expression levels correlating with poor clinical outcomes in human breast cancer patients . ALYREF has been demonstrated to significantly influence cellular growth in triple-negative breast cancer (TNBC) cells and tumor formation in vivo, suggesting its potential as both a prognostic biomarker and therapeutic target . Additionally, ALYREF has been shown to drive cancer cell proliferation in glioblastoma through an ALYREF-MYC positive feedback loop mechanism .

Which experimental applications are most suitable for ALYREF antibodies?

ALYREF antibodies are versatile tools suitable for multiple experimental applications:

These applications allow researchers to detect ALYREF protein expression, localization, and interactions in various experimental contexts .

How do I optimize ALYREF antibody use for Western blotting experiments?

For optimal Western blotting results with ALYREF antibodies:

• Be aware that while the calculated molecular weight of ALYREF is 26 kDa, the observed band typically appears at approximately 30 kDa . This discrepancy is normal and likely due to post-translational modifications.

• Use appropriate blocking buffers (typically 5% non-fat milk or BSA in TBST) to minimize background signal.

• Include positive control samples such as HeLa or A549 cell lysates, which have been verified to express detectable levels of ALYREF .

• For challenging samples, consider longer exposure times or signal enhancement systems, as ALYREF expression levels may vary across tissue types.

• When interpreting results, remember that ALYREF has multiple modified forms which may result in additional bands on the membrane .

What is the subcellular localization of ALYREF and how does this impact immunofluorescence experiments?

ALYREF exhibits a complex subcellular distribution pattern that should be considered when designing immunofluorescence experiments. It is primarily localized in the nucleus and nuclear speckles, but can also be found in the cytoplasm . ALYREF colocalizes with the exon junction complex (EJC), THOC4, NXF1, and DDX39B in the nucleus and nuclear speckles, and travels to the cytoplasm as part of the EJC bound to mRNA .

For successful immunofluorescence experiments:

• Use appropriate nuclear counterstains (such as DAPI) to verify nuclear localization.

• Consider co-staining experiments with markers of nuclear speckles or EJC components to confirm functional localization.

• Optimize fixation and permeabilization methods to preserve both nuclear and potential cytoplasmic signals.

• Use confocal microscopy when possible to better resolve the nuclear speckle pattern characteristic of ALYREF.

How can I investigate ALYREF's role in mRNA processing and export using antibody-based approaches?

ALYREF plays a crucial role in both polyadenylated and non-polyadenylated mRNA metabolism pathways, coordinating processing and nuclear export . To investigate these functions:

• RNA Immunoprecipitation (RIP) assay: Use ALYREF antibodies to immunoprecipitate ALYREF-bound RNAs followed by RT-qPCR analysis. This approach has successfully demonstrated ALYREF binding to histone mRNAs . Protocol considerations include:

  • RNase inhibitor addition is critical throughout the procedure

  • Crosslinking optimization (UV crosslinking at 254 nm is often used)

  • Stringent washing conditions to reduce background

• iCLIP-seq (individual-nucleotide-resolution UV crosslinking and immunoprecipitation and sequencing): This technique has revealed that ALYREF universally binds to regions next to the stem-loop on replication-dependent histone mRNAs . Implementation requires:

  • Specialized crosslinking equipment

  • Library preparation optimization

  • Bioinformatic pipeline for data analysis

• Co-immunoprecipitation: To study protein-protein interactions, as demonstrated in investigating ALYREF association with SLBP (stem-loop binding protein). Research has shown that ALYREF associates with SLBP through protein-protein interaction .

What experimental approaches can demonstrate the functional significance of ALYREF in cancer progression?

To investigate ALYREF's functional role in cancer:

• Knockdown/knockout experiments: siRNA-mediated knockdown or CRISPR-based knockout of ALYREF has been shown to significantly reduce cellular growth, colony formation, mammosphere formation, and anchorage-independent growth in triple-negative breast cancer cell lines . Similar approaches in glioblastoma cells demonstrated that ALYREF inhibition dramatically downregulated cell proliferation and abolished tumorigenicity in vivo .

• Overexpression studies: Stable overexpression of ALYREF in SUM159 cells led to enhanced cellular growth, increased colony formation, more colonies in soft agar assays, and increased mammosphere formation . This complementary approach confirms the oncogenic potential of ALYREF.

• In vivo tumor formation assays: Inducible knockdown of ALYREF expression in orthotopic breast tumors significantly decreased tumor formation, indicating therapeutic potential . For glioblastoma, xenograft experiments using CRISPR to target ALYREF showed dramatically abolished tumorigenicity .

• Molecular mechanism studies: In glioblastoma, CLIP-qPCR analysis demonstrated that ALYREF binds to MYC mRNA, specifically in the 3′-UTR region, controlling its stability . 3′-UTR reporter assays and mRNA stability assays confirmed this mechanism.

How can I address technical challenges when working with ALYREF antibodies in tissue samples?

Researchers frequently encounter challenges when working with ALYREF antibodies in tissue samples:

• Variable expression levels: ALYREF expression varies significantly between cancer subtypes and even within the same cancer type. For example, triple-negative breast cancers typically show higher expression than other subtypes . Consider:

  • Including multiple positive and negative control tissues

  • Using amplification steps for low-expressing samples

  • Optimizing antigen retrieval methods for specific tissue types

• Background signal: Nuclear proteins can exhibit high background staining. To minimize this:

  • Test several blocking buffers (BSA, normal serum, commercial blockers)

  • Optimize antibody concentration through titration experiments

  • Increase washing steps or duration

  • Consider using monoclonal antibodies if polyclonal antibodies show high background

• Specificity verification: Always validate antibody specificity through:

  • Peptide competition assays

  • Comparison with RNA expression data

  • Use of ALYREF-knockout tissues or cells as negative controls

  • Western blot verification of a single band at approximately 30 kDa

What considerations are important when designing experiments to study ALYREF in different cancer types?

When investigating ALYREF across cancer types, consider:

• Cancer-specific expression patterns: Analysis of large patient cohorts (METABRIC, TCGA-PanCancer Atlas, INSERM, MBC project) has demonstrated variable ALYREF amplification and expression patterns across cancer types . Design experiments to account for:

  • Tissue-specific expression levels

  • Cancer subtype variations

  • Correlation with other biomarkers

• Context-dependent molecular partners: ALYREF interacts with different molecular partners depending on cancer type. In breast cancer, its interactions with transcriptional machinery are critical , while in glioblastoma, its binding to MYC mRNA is crucial for oncogenesis . Consider:

  • Co-immunoprecipitation experiments targeting tissue-specific interacting partners

  • Proximity ligation assays to verify protein-protein interactions in situ

  • RNA-IP followed by sequencing to identify cancer-specific RNA targets

• Functional readouts: Different cancer types may require different functional assays:

  • Soft agar colony formation works well for some cell lines (SUM159) but not others (BT-549, MDA-MB-468)

  • Proliferation, migration, invasion, and stem cell assays should be selected based on the cancer's specific biological characteristics

How should I interpret inconsistencies between ALYREF antibody results and mRNA expression data?

Discrepancies between protein and mRNA expression levels are common and may reflect important biological mechanisms:

• Post-transcriptional regulation: ALYREF itself regulates mRNA processing and export, creating potential feedback loops. Research has shown that ALYREF can bind to MYC mRNA and enhance its stability in GBM cells , suggesting that similar mechanisms may apply to other mRNAs.

• Post-translational modifications: ALYREF undergoes modifications that affect its stability and function. The observed molecular weight (30 kDa) differs from the calculated weight (26 kDa) , indicating modifications that may vary between tissues or disease states.

• Localization changes: As ALYREF shuttles between the nucleus and cytoplasm , subcellular fractionation experiments may be necessary to determine whether apparent expression changes reflect redistribution rather than absolute quantity changes.

• Technical considerations: When encountering discrepancies:

  • Verify antibody specificity using knockdown/knockout controls

  • Test multiple antibodies targeting different epitopes

  • Use absolute quantification methods like quantitative Western blotting with recombinant protein standards

What are the critical controls needed when studying ALYREF in co-immunoprecipitation experiments?

When performing co-immunoprecipitation to study ALYREF interactions:

• RNase treatment control: Since ALYREF is an RNA-binding protein, determine whether interactions are RNA-dependent by comparing results with and without RNase A treatment, as demonstrated in studies of ALYREF-SLBP interaction .

• Antibody specificity controls:

  • IgG control immunoprecipitation must be performed in parallel

  • Input samples (pre-IP lysate) should be analyzed to confirm target protein expression

  • Reverse co-IP (immunoprecipitating the suspected interacting partner and blotting for ALYREF) should confirm results

• Competition controls: For validation, consider peptide competition or expression of truncated protein domains to map interaction interfaces.

• Crosslinking considerations: For transient or weak interactions, chemical crosslinking may be necessary but requires careful optimization to avoid artifacts.

How can I reconcile contradictory findings about ALYREF function in different experimental systems?

Research has revealed that ALYREF may have context-dependent functions:

• Cancer type specificity: While ALYREF is upregulated and promotes proliferation in breast cancer and glioblastoma , it has been reported as downregulated in skin and testicular cancers . When analyzing contradictory results:

  • Consider tissue-specific transcriptional programs

  • Evaluate experimental models (cell lines vs. primary cells vs. tissues)

  • Examine genetic background differences between experimental systems

• Functional redundancy: ALYREF belongs to the TREX complex, and other components may compensate for its loss in certain contexts. Studies show that UAP56 and THO components can also be detected on histone mRNAs , suggesting potential functional overlap.

• Direct vs. indirect effects: Distinguish between direct ALYREF functions and secondary effects:

  • Use acute vs. chronic depletion systems

  • Employ rescue experiments with wild-type and mutant ALYREF

  • Conduct time-course experiments to establish causality

How can ALYREF antibodies be used to investigate its potential as a therapeutic target in cancer?

ALYREF's role in promoting cancer cell proliferation makes it an intriguing therapeutic target:

• Target validation approaches:

  • Inducible knockdown systems in established tumors have demonstrated that reducing ALYREF expression can decrease tumor formation in vivo

  • CRISPR-mediated knockout of ALYREF dramatically abolished tumorigenicity in xenograft models

  • These findings indicate strong potential for therapeutic interventions targeting ALYREF

• Biomarker development:

  • Immunohistochemistry with validated ALYREF antibodies could stratify patients according to expression levels

  • Survival analyses from two independent breast cancer cohorts suggest ALYREF as a novel prognostic biomarker

  • Correlation studies between ALYREF and MYC expression could identify patients who might benefit from targeted therapies

• Therapeutic resistance monitoring:

  • Monitor ALYREF expression changes during treatment using validated antibodies

  • Investigate whether ALYREF-mediated mRNA export contributes to therapeutic resistance by regulating stress response genes

What methodological approaches can elucidate the role of ALYREF in regulating specific mRNA subsets in cancer?

To investigate ALYREF's selective regulation of mRNAs:

• Integrated RNA-binding and functional analyses:

  • Combine CLIP-seq to identify binding sites with RNA-seq following ALYREF manipulation

  • This approach identified ALYREF binding to MYC mRNA 3′-UTR in GBM cells

  • Similar strategies could identify cancer-specific ALYREF RNA targets

• mRNA stability assays:

  • Measure decay rates of candidate mRNAs after transcriptional inhibition

  • ALYREF knockdown reduced MYC mRNA stability in GBM cells , suggesting this approach can identify direct regulatory targets

• Reporter assays with mutated binding sites:

  • 3′-UTR reporter plasmids can assess ALYREF's direct effect on mRNA stability

  • Mutation of predicted binding sites can confirm specificity of regulation

  • This approach confirmed ALYREF regulation of MYC via its 3′-UTR

How can multi-omics approaches incorporating ALYREF antibody-based techniques advance our understanding of cancer biology?

Integrating ALYREF studies with multi-omics approaches offers powerful insights:

• Proteogenomic integration:

  • Combine ALYREF ChIP-seq, CLIP-seq, and proteomics of ALYREF-interacting proteins

  • This could reveal how genomic alterations of ALYREF (observed in large cancer cohorts ) affect its protein interactions and RNA targets

  • Such integration could identify context-specific regulatory networks

• Single-cell analyses:

  • Apply ALYREF antibodies in single-cell imaging mass cytometry or multiplexed immunofluorescence

  • This would reveal intratumoral heterogeneity of ALYREF expression and localization

  • Correlation with other cancer markers at single-cell resolution could identify previously unrecognized cellular states

• Spatial transcriptomics with protein validation:

  • Combine spatial transcriptomics with ALYREF immunohistochemistry in sequential sections

  • This would map relationships between ALYREF protein expression and spatially resolved transcriptomes

  • Such approaches could reveal tumor microenvironment influences on ALYREF function

What factors should be considered when selecting ALYREF antibodies for specific research applications?

When selecting ALYREF antibodies:

• Epitope location: Consider where the antibody binds within the ALYREF protein structure:

  • N-terminal antibodies may detect all isoforms

  • Antibodies targeting functional domains may be blocked when ALYREF is in protein complexes

  • The immunogen used (recombinant protein of human ALYREF ) determines epitope availability

• Cross-reactivity: Verify species reactivity for your experimental system:

  • Available antibodies show reactivity with human, mouse, and rat ALYREF

  • Sequence conservation analysis is recommended when working with other species

• Application-specific validation:

  • For Western blotting: Verify single band at approximately 30 kDa

  • For IHC: Confirm appropriate nuclear/nuclear speckle staining pattern

  • For IF: Validate colocalization with nuclear speckle markers

• Monoclonal vs. polyclonal considerations:

  • Polyclonal antibodies provide signal amplification but may have batch variation

  • Monoclonal antibodies offer consistency but may be more sensitive to epitope masking

How can researchers effectively study dynamic interactions between ALYREF and its molecular partners?

To capture dynamic ALYREF interactions:

• Live-cell imaging approaches:

  • Fluorescent protein tagging of ALYREF combined with potential interaction partners

  • FRET/BRET systems to detect proximity in living cells

  • Photobleaching techniques to measure binding kinetics

• Sequential immunoprecipitation strategies:

  • Studies have shown that ALYREF associates with SLBP through protein-protein interaction

  • Two-step immunoprecipitation can identify complexes containing multiple components

  • Time-course experiments following stimulation can capture interaction dynamics

• Proximity labeling technologies:

  • BioID or APEX2 fused to ALYREF can identify proximal proteins in living cells

  • Temporal control of labeling enables the study of interaction changes during cellular processes

  • This approach could extend findings on ALYREF's role in mRNA export complexes

What experimental design is optimal for investigating ALYREF's dual role in mRNA processing and export?

ALYREF coordinates both processing and nuclear export of mRNAs . To dissect these functions:

• Function-specific mutants:

  • Design ALYREF mutants that selectively disrupt specific protein interactions

  • Test their impact on discrete steps of mRNA processing and export

  • Use these mutants in rescue experiments following endogenous ALYREF depletion

• Sequential process analysis:

  • Nuclear/cytoplasmic fractionation to track mRNA movement

  • Nascent RNA capture to study ALYREF's role during transcription

  • RNA processing assays to evaluate 3′-end formation efficiency

• Structured experimental approaches:

  • First establish ALYREF binding to target mRNAs (e.g., histone mRNAs )

  • Then determine effects on processing (e.g., 3′-end formation)

  • Finally assess export efficiency through subcellular distribution analysis