CELF1 Antibody

What is CELF1 and what cellular functions does it perform?

CELF1 is a highly conserved RNA-binding protein that regulates multiple aspects of RNA metabolism. It functions in the nucleus to mediate alternative splicing and in the cytoplasm to regulate mRNA stability and translation by binding to GU-rich elements (GREs) . CELF1 was originally identified as a protein that binds to CUG triplet repeats within RNA and has since been implicated in various developmental processes and disease states, including myotonic dystrophy type 1 (DM1), dilated cardiomyopathy, tumor metastasis, and liver fibrosis . Recent studies have established CELF1 as a central node in post-transcriptional regulatory networks, particularly in processes like epithelial-to-mesenchymal transition (EMT) during cancer progression .

Where is CELF1 protein localized within cells and tissues?

CELF1 exhibits dynamic subcellular localization that reflects its diverse functions:

• Nuclear localization: Immunohistochemistry studies using anti-CELF1 antibodies show specific staining in nuclei of various tissues, including human skeletal muscle, where it regulates pre-mRNA splicing .

• Cytoplasmic distribution: In the cytoplasm, CELF1 binds to GRE-containing transcripts to regulate their stability and translation efficiency .

• Stress granules: Under cellular stress conditions, CELF1 has been observed to localize to cytoplasmic stress granules .

This distribution pattern can shift during development and in disease states. For instance, in myotonic dystrophy type 1 (DM1), abnormal nuclear accumulation of CELF1 leads to mRNA dysregulation .

What RNA sequences does CELF1 preferentially bind to?

CELF1 demonstrates specific RNA binding preferences that have been characterized through various experimental approaches:

• GU-rich elements (GREs): CELF1 predominantly binds to GU-rich motifs in the 3′-UTR of target mRNAs . These interactions are critical for post-transcriptional regulation of gene expression.

• Exon-intron boundaries: RIP-seq experiments in HeLa cells revealed that CELF1 preferentially binds at exon-intron boundaries, with a biased distribution at 3'UTR and intronic regions .

• Splice site motifs: Bioinformatic analyses of CELF1-bound regions show enrichment for both 5' and 3' splice site motifs, supporting its role in regulating alternative splicing .

The binding specificity is mediated by CELF1's three RNA-recognition motifs (RRMs), with specific residues within these domains being particularly important for RNA interactions .

How is CELF1 expression regulated during development?

CELF1 expression and activity are dynamically regulated during development in a tissue-specific manner:

• Alternative UTR regulation: The distribution of CELF1 3′UTR isoforms is tightly controlled during skeletal muscle, heart, and brain development . This regulation affects protein expression by altering sensitivity to specific microRNAs or RNA-binding proteins.

• Alternative 5′UTR processing: Alternative splicing of the CELF1 5′UTR leads to translation of distinct protein isoforms with different N-terminal domain lengths .

• Post-translational control: CELF1 protein undergoes post-translational modifications during developmental programs like EMT, affecting its activity independent of mRNA level changes .

Disruption of normal CELF1 regulation occurs in certain pathological contexts, such as myotonic dystrophy type 1, where aberrant processing of CELF1 mRNA contributes to disease progression .

What technical specifications should researchers know when using CELF1 antibodies?

When working with CELF1 antibodies, researchers should consider these technical specifications:

• Molecular weight: CELF1 protein typically appears as a specific band at approximately 50 kDa when detected by Western blot .

• Optimal concentrations: For Western blot applications, 0.5 μg/mL antibody concentration is often sufficient; for immunohistochemistry on paraffin sections, 15 μg/mL has been effective .

• Cross-reactivity: Validated antibodies like clone 850717 recognize human, mouse, and rat CELF1, facilitating comparative studies across species .

• Application versatility: CELF1 antibodies have been successfully used in multiple applications including Western blotting, immunohistochemistry, and immunoprecipitation experiments .

The choice of antibody should be guided by the specific research application and experimental conditions.

How can researchers validate CELF1 antibody specificity for RNA immunoprecipitation?

For reliable RNA immunoprecipitation (RIP) experiments with CELF1, thorough antibody validation is essential:

Recommended validation protocol:

• Western blot assessment:

  • Test the antibody on lysates from cells known to express CELF1 (e.g., HeLa, Neuro-2A)

  • Confirm detection of a single specific band at ~50 kDa

  • Include CELF1 knockdown samples as negative controls

• IP-Western validation:

  • Perform immunoprecipitation using the CELF1 antibody

  • Analyze immunoprecipitated material by Western blot

  • Probe with a different CELF1 antibody targeting a distinct epitope

• Functional validation:

  • Assess enrichment of known CELF1 target RNAs in immunoprecipitates

  • Compare results using multiple anti-CELF1 antibodies

  • Include non-target RNAs (e.g., ACTB mRNA has been used as a negative control)

Research indicates that using two distinct anti-CELF1 antibodies for immunoprecipitation provides robust validation, as demonstrated in studies with epithelial and mesenchymal MCF10A cells .

What experimental approaches are most effective for studying CELF1-RNA interactions?

Multiple complementary techniques provide comprehensive insights into CELF1-RNA interactions:

RIP-seq (RNA Immunoprecipitation-sequencing)

• Identifies genome-wide RNA targets

• Has revealed biased distribution of CELF1 binding at 3'UTR and intronic regions

• RIP-seq in HeLa cells identified 15,285 CELF1-specific sense peaks enriched for splice site motifs and GU-rich elements

UV Crosslinking/Immunoprecipitation/qRT-PCR

• Preserves direct physical interactions between CELF1 and RNA

• Allows quantitative assessment of binding to specific targets

• Successfully demonstrated enrichment of GRE-containing mRNAs in CELF1 immunoprecipitates from mesenchymal cells

Reporter Assays with Wild-Type vs. Mutated Binding Sites

• Directly tests the functionality of putative binding sites

• Compare wild-type 3′-UTRs vs. 3′-UTRs with deleted GRE elements (ΔGRE)

• Studies showed markedly diminished enrichment of ΔGRE reporters compared to wild-type controls in TGF-β-treated cells

These approaches are most powerful when combined to establish both binding patterns and functional significance of CELF1-RNA interactions.

How does CELF1 function differ between epithelial and mesenchymal cellular states?

CELF1 exhibits distinct activity patterns in epithelial versus mesenchymal cellular contexts:

In Epithelial Cells:

• Lower CELF1 binding activity to GRE-containing mRNAs

• Immunoprecipitation with anti-CELF1 antibodies shows minimal enrichment of GRE-containing transcripts

• CELF1-RNA interactions are less prominent in maintaining epithelial phenotype

In Mesenchymal Cells:

• Significantly increased binding to GRE-containing mRNAs encoding EMT drivers

• Enhanced translation of these target transcripts

• TGF-β treatment further promotes CELF1-RNA interactions

Research has established that CELF1's ability to drive EMT depends on its RNA-binding functionality. RNA-binding mutants of CELF1 (with alterations in key residues of the RRMs) fail to enrich GRE-containing mRNAs and cannot promote mesenchymal transition . This differential activity makes CELF1 a central node in post-transcriptional regulatory programs underlying EMT and tumor progression.

What role does CELF1 play in alternative splicing regulation?

CELF1 functions as a key regulator of alternative splicing through specific mechanisms:

• Exon-intron boundary binding: CELF1 preferentially binds at exon-intron boundaries, influencing splice site selection .

• Global splicing effects: Transcriptome analyses reveal that alternative splicing is globally regulated by CELF1 in multiple cell types .

• Specific exon regulation: For example, CELF1 positively regulates inclusion of exon 16 of the LMO7 gene, a marker gene for breast cancer .

• Developmental splicing programs: The distribution of CELF1 isoforms themselves is regulated during skeletal muscle, heart, and brain development through alternative splicing .

Experimental approaches to study CELF1's splicing function include:

• CELF1 depletion/overexpression followed by transcriptome analysis

• Minigene splicing assays with target exons

• Binding site mutagenesis to disrupt CELF1 recognition

• Integration of binding data with splicing outcomes to construct RNA-maps

These methodologies have revealed that CELF1 recognizes both 5' and 3' splice site motifs as well as GU-rich elements, allowing it to influence multiple aspects of pre-mRNA processing .

How can small molecule inhibitors of CELF1 be utilized in research?

Small molecules targeting CELF1 provide valuable research tools:

Available compounds and mechanisms:

• Compound 27: Disrupts CELF1-RNA binding by competing with RNA for binding to CELF1

• Compound 841: A derivative of compound 27, identified as a selective CELF1 inhibitor through structure-activity relationship analysis

Research applications:

• Mechanism dissection: Distinguish between RNA-binding dependent and independent functions of CELF1

• Temporal studies: Apply compounds at different time points to determine critical windows for CELF1 activity

• Therapeutic potential assessment: Evaluate compounds in disease models (e.g., liver fibrosis, myotonic dystrophy)

Experimental evidence:
Compound 27 promotes IFN-γ secretion and suppresses TGF-β1-induced hepatic stellate cell activation by inhibiting CELF1-mediated IFN-γ mRNA decay . In vivo, this compound attenuates CCl₄-induced murine liver fibrosis, demonstrating that targeting CELF1 RNA-binding activity with small molecules represents a viable approach for treating CELF1-mediated diseases .

How is CELF1 dysregulated in cancer progression?

CELF1 exhibits specific patterns of dysregulation in cancer that can be detected using appropriate antibodies:

• Protein overexpression: CELF1 protein, but not mRNA, is significantly overexpressed in human breast cancer tissues, suggesting post-transcriptional regulation .

• Functional significance: CELF1 functions as a central node controlling translational activation of genes driving epithelial-to-mesenchymal transition (EMT) .

• Mechanistic pathway: An 11-component genetic pathway has been identified in which CELF1 controls translational activation of EMT drivers through binding to GU-rich elements in their 3'UTRs .

Research has established that CELF1 is both necessary and sufficient for mesenchymal transition and metastatic colonization . This makes it a potential biomarker and therapeutic target in cancer progression.

What is CELF1's role in myotonic dystrophy pathogenesis?

In myotonic dystrophy type 1 (DM1), CELF1 dysfunction plays a central role:

• Altered regulation: A CTG repeat expansion mutation in the 3′UTR of DM protein kinase (DMPK) leads to nuclear accumulation of CELF1 .

• Splicing dysregulation: Abnormal CELF1 activity causes widespread mRNA splicing defects .

• CELF1 isoform changes: The distribution of CELF1 3′UTR isoforms is disrupted in skeletal muscles in the context of DM1 .

These alterations contribute to the complex pathology of myotonic dystrophy, affecting multiple tissues including skeletal muscle, heart, and brain. CELF1 antibodies have been crucial in elucidating these mechanisms by enabling detection of altered CELF1 expression and localization patterns in patient samples.

How can CELF1 antibodies be utilized to study liver fibrosis mechanisms?

CELF1 plays important roles in liver fibrosis pathogenesis that can be investigated using specific antibodies:

• Regulatory pathway: CELF1 regulates IFN-γ mRNA decay, affecting hepatic stellate cell (HSC) activation during fibrosis .

• Intervention approach: Small molecules targeting CELF1 RNA-binding activity (e.g., compound 27) attenuate liver fibrosis by inhibiting CELF1-mediated IFN-γ mRNA decay .

• Experimental applications:

  • Immunohistochemistry to track CELF1 expression in fibrotic liver tissues

  • RNA immunoprecipitation to identify CELF1 targets during disease progression

  • Western blotting to monitor changes in CELF1 levels during therapeutic interventions

These applications of CELF1 antibodies help elucidate the molecular mechanisms underlying liver fibrosis and assess potential therapeutic strategies targeting this RNA-binding protein.

What controls should be included in CELF1 antibody-based experiments?

Robust controls are essential for generating reliable data with CELF1 antibodies:

Negative Controls:

• Isotype control antibodies: Use isotype-matched IgG for immunoprecipitation and staining experiments

• Non-target RNA controls: Include RNAs not bound by CELF1 (e.g., ACTB mRNA has been validated)

• CELF1-depleted samples: Use siRNA knockdown or CRISPR knockout cells

• Binding site mutants: Compare wild-type sequences to those with mutated CELF1 binding sites

Positive Controls:

• Known CELF1 targets: Include established targets (e.g., GRE-containing EMT drivers)

• Cell types with confirmed expression: HeLa, Neuro-2A, and Rat-2 cell lines consistently express detectable CELF1

• Recombinant CELF1 protein: Include purified protein for antibody validation

Functional Controls:

• RNA-binding mutants: Use CELF1 variants with mutations in RNA recognition motifs (RRMs)

• Domain deletion mutants: Test constructs lacking specific functional domains

In published studies, researchers have validated CELF1-RNA interactions by comparing enrichment of wild-type 3′-UTRs versus 3′-UTRs with deleted GRE elements (ΔGRE), observing significantly diminished enrichment of mutant constructs .

How can researchers distinguish between CELF1 isoforms?

Distinguishing between CELF1 isoforms requires strategic antibody selection and experimental design:

CELF1 isoform characteristics:

• Alternative splicing of the CELF1 5′UTR leads to translation of multiple protein isoforms with different N-terminal domains

• The distribution of CELF1 3′UTR isoforms is developmentally regulated in tissue-specific patterns

Antibody selection strategies:

• Epitope-specific antibodies:

  • Choose antibodies targeting regions unique to specific isoforms

  • Review immunogen information (e.g., antibody clone 850717 targets Met1-Gly60 of human CELF1)

• Experimental approach:

  • Use high-resolution gels to separate closely migrating isoforms

  • Compare banding patterns between tissues known to express different isoform ratios

  • Consider 2D gel electrophoresis for isoforms with similar weights but different modifications

Research has demonstrated that CELF1 isoforms with different 5′ and 3′ UTRs show distinct patterns of expression during skeletal muscle, heart, and brain development, with disruption of these patterns in DM1 .

What are the comparative advantages of different detection methods for CELF1?

The choice of detection method should be guided by specific research questions:

Western Blotting:

• Advantages: Quantitative assessment of protein levels; detects post-translational modifications

• Limitations: Lacks spatial information within cells/tissues

• Optimization: Use 0.5 μg/mL antibody concentration; a specific band should be detected at ~50 kDa

Immunohistochemistry/Immunofluorescence:

• Advantages: Provides spatial localization within cells and tissues

• Limitations: Semi-quantitative; fixation conditions may affect epitope accessibility

• Example application: CELF1 has been successfully detected in human skeletal muscle using 15 μg/mL antibody concentration, showing specific nuclear localization

RNA Immunoprecipitation (RIP):

• Advantages: Identifies RNA targets bound by CELF1; can be coupled with sequencing

• Limitations: Requires careful validation and controls

• Application note: Successfully applied to identify thousands of CELF1-bound RNAs in HeLa cells, revealing preferential binding at 3'UTR and intronic regions

For comprehensive characterization, combining multiple detection approaches provides the most complete understanding of CELF1 biology in experimental systems.

What emerging technologies might enhance CELF1 research?

Emerging technologies poised to advance CELF1 research include:

• CRISPR-based RNA targeting: Systems like CRISPR-Cas13 could allow precise manipulation of CELF1-RNA interactions in living cells

• Single-molecule imaging: Techniques to visualize individual CELF1-RNA complexes in real-time within cells

• Spatial transcriptomics: Methods to map CELF1-regulated RNA processing events within tissues with spatial resolution

• Small molecule library screening: High-throughput approaches to identify additional compounds targeting CELF1 with improved specificity

• Cryo-EM structural analysis: Detailed structural characterization of CELF1-RNA complexes to inform rational drug design

These technologies promise to deepen our understanding of CELF1 biology and accelerate therapeutic development for CELF1-mediated diseases.

How might CELF1 research translate to clinical applications?

CELF1 research shows promising pathways for clinical translation:

• Diagnostic biomarkers: CELF1 protein overexpression in breast cancer could serve as a prognostic indicator

• Therapeutic targeting: Small molecules like compound 27 and compound 841 that disrupt CELF1-RNA binding represent potential treatments for liver fibrosis and potentially other CELF1-mediated conditions

• RNA-based therapeutics: Antisense oligonucleotides or siRNAs targeting CELF1 or its regulatory pathways

• Combination therapies: Targeting CELF1 alongside other disease mechanisms for synergistic effects

The central role of CELF1 in post-transcriptional regulation across multiple diseases positions it as a valuable target for developing novel therapeutic strategies.