CELF6 Antibody

What is CELF6 and why is it significant in molecular biology research?

CELF6 (CUGBP Elav-Like Family Member 6) is an RNA-binding protein that belongs to the cytosine-uridine-guanine-binding protein (CUG-BP), Elav-like family (CELF). It plays critical roles in post-transcriptional regulation of pre-mRNAs through binding to specific RNA sequences . The significance of CELF6 in research stems from its multifunctional roles in:

• Regulating mRNA stability and processing in various cellular contexts

• Cell cycle regulation through modulation of p21 gene expression and mRNA stability

• Cancer development, with evidence suggesting tumor suppressor functions in colorectal and lung cancers

• Neurobiological processes, particularly in serotonergic neurons, with implications for neuropsychiatric disorders including autism

Understanding CELF6 biology has become increasingly important due to its downregulation in several cancer types and its association with poor clinical outcomes, positioning it as both a potential biomarker and therapeutic target .

What are the validated applications for CELF6 antibodies in research settings?

CELF6 antibodies have been validated for multiple research applications, each requiring specific protocols and considerations:

Research has demonstrated that antibodies generated against the QPGSDTLYNNGVSPC peptide sequence are effective for both Western blot and immunofluorescence applications, while antibodies against AASEGRGEDRKC peptide are only effective for Western blot, highlighting the importance of selecting appropriate antibodies for specific applications .

How should researchers validate CELF6 antibodies before experimental use?

Thorough validation of CELF6 antibodies is essential for generating reliable data. A comprehensive validation protocol should include:

• Specificity testing:

  • Western blot analysis showing a single band at the expected molecular weight (~50-60 kDa)

  • Comparison with known positive and negative controls

  • Testing in cells transfected with GFP-tagged and untagged CELF6 isoforms

  • Validation in CELF6 knockout or knockdown models if available

• Application-specific validation:

  • For immunofluorescence: Testing fixation conditions (4% paraformaldehyde has been validated)

  • For Western blot: Optimizing blocking conditions (5% non-fat milk in TBST is effective)

  • For all applications: Testing antibody performance across multiple cell types and tissues

• Experimental controls:

  • Include GAPDH or β-actin as loading controls for Western blots

  • Use isotype controls for immunoprecipitation experiments

  • Include secondary antibody-only controls for immunostaining procedures

Researchers should be aware that not all CELF6 antibodies work equally well across different applications, necessitating application-specific validation .

What are the optimal conditions for using CELF6 antibodies in Western blotting?

Optimizing Western blot protocols for CELF6 detection requires attention to several technical parameters:

• Sample preparation:

  • Use complete lysis buffers containing protease inhibitors

  • Standard RIPA or NP-40 buffers are generally effective for CELF6 extraction

  • Consider phosphatase inhibitors if studying post-translational modifications

• Gel electrophoresis and transfer:

  • 10% SDS-PAGE gels provide appropriate resolution for CELF6

  • Standard PVDF membranes are suitable for CELF6 detection

  • Transfer efficiency can be verified using reversible protein stains

• Antibody incubation:

  • Blocking: 5% non-fat milk in TBST for 1 hour at room temperature

  • Primary antibody: 1:1,000 dilution of CELF6 antibody (overnight at 4°C preferred)

  • Secondary antibody: HRP-conjugated anti-rabbit at 1:10,000 dilution

• Detection:

  • Enhanced chemiluminescence (ECL) reagents provide suitable sensitivity

  • Exposure times may need optimization depending on CELF6 expression levels

  • Digital imaging systems allow precise quantification

When comparing CELF6 expression between samples, normalization to housekeeping proteins such as GAPDH is essential for accurate quantification .

How can researchers effectively study CELF6's role in RNA regulation?

CELF6 functions as an RNA-binding protein that modulates mRNA stability. To investigate this function, researchers should consider these methodological approaches:

• RNA immunoprecipitation (RIP) assays:

  • Immunoprecipitate CELF6 using validated antibodies

  • Extract and analyze associated RNAs through RT-PCR or RNA-seq

  • This approach has successfully identified p21 mRNA as a CELF6 target

• mRNA stability assays:

  • Treat cells with transcription inhibitors (e.g., actinomycin D)

  • Monitor decay rates of potential target mRNAs in CELF6-overexpressing versus control cells

  • This approach demonstrated that CELF6 increases p21 mRNA stability

• Binding site identification:

  • In vitro binding assays with recombinant CELF6 and target RNA sequences

  • Mutational analysis of putative binding sites

  • Cross-linking immunoprecipitation (CLIP) methods to identify binding sites with high resolution

• Functional validation:

  • CELF6 overexpression or knockdown experiments

  • Assessment of target mRNA and protein levels

  • Analysis of downstream cellular phenotypes

Studies have demonstrated that CELF6 binds to the 3′UTR of p21 transcript, increasing its mRNA stability, which contributes to cell cycle regulation and potential tumor suppressor activity .

What experimental models are most appropriate for studying CELF6 function?

Selecting appropriate experimental models is crucial for CELF6 research. Current evidence supports the following models:

• Cell line models:

  • Colorectal cancer cell lines: Demonstrate CELF6 downregulation and effects on cell proliferation and stemness

  • Lung cancer cell lines (A549): Show transcriptional changes upon CELF6 overexpression

  • 3T3 cells: Useful for antibody validation studies with transfected CELF6 constructs

• Animal models:

  • CELF6 knockout mice: Generated by deleting exon 4 of the Celf6 gene using Cre-lox recombination

  • Useful for studying neuropsychiatric phenotypes and validating antibody specificity

• Patient-derived samples:

  • Tissue microarrays comparing tumor vs. normal tissues for CELF6 expression

  • Cancer patient cohorts for correlation with clinical outcomes

• Genetic models:

  • CRISPR/Cas9-mediated CELF6 knockout or mutation

  • Inducible overexpression systems for temporal control of CELF6 expression

Each model offers distinct advantages depending on the research question. For cancer-related studies, both in vitro cell culture and in vivo xenograft models have provided valuable insights into CELF6's tumor suppressor functions .

How can CELF6 antibodies be used to investigate its role in colorectal cancer?

CELF6 has emerged as a potential tumor suppressor in colorectal cancer (CRC). Researchers can employ CELF6 antibodies in several sophisticated approaches:

• Expression analysis in clinical samples:

  • Immunohistochemistry of tissue microarrays to correlate CELF6 expression with clinical stages and outcomes

  • Western blot analysis comparing normal colonic mucosa versus CRC tissues at various stages

• Mechanistic investigations:

  • Co-immunoprecipitation to identify CELF6 protein interaction partners in CRC cells

  • ChIP-seq or RIP-seq to identify cancer-specific genomic or RNA targets

  • Analysis of CELF6-mediated regulation of HOXA5 mRNA stability

• Functional analysis:

  • Immunofluorescence to track changes in CELF6 localization during cell cycle progression

  • Western blot analysis to monitor CELF6 expression after drug treatments or genetic manipulations

• Cancer stem cell studies:

  • Analysis of CELF6 expression in cancer stem cell populations using flow cytometry and immunofluorescence

  • Investigation of CELF6's role in regulating stemness-related genes

Research has demonstrated that CELF6 overexpression decreases CRC cell proliferation and stemness in vitro, reduces tumor growth in vivo, and induces G1 phase cell cycle arrest . These findings position CELF6 as a promising target for therapeutic development in CRC.

What methods can be used to study the relationship between CELF6 and cell cycle regulation?

CELF6 has been identified as a cell cycle-regulated protein that controls cancer cell proliferation through p21 regulation. Researchers should consider these methodological approaches:

• Cell cycle synchronization and analysis:

  • Synchronize cells at different cell cycle phases

  • Analyze CELF6 protein levels by Western blot across cell cycle stages

  • Immunofluorescence to visualize CELF6 localization changes during cell cycle progression

• Degradation pathway analysis:

  • Investigate ubiquitin-proteasome-mediated regulation of CELF6

  • Study SCF-β-TrCP recognition of CELF6 through nonphospho motifs

  • Treatment with proteasome inhibitors to confirm degradation mechanisms

• Downstream effect analysis:

  • Flow cytometry for cell cycle distribution after CELF6 overexpression or knockdown

  • BrdU incorporation assays to measure proliferation

  • Colony formation assays to assess long-term proliferative capacity

• p21-focused experiments:

  • Luciferase reporter assays with p21 3'UTR constructs

  • mRNA stability assays comparing p21 mRNA half-life in CELF6-modulated cells

  • Rescue experiments with p21 knockdown in CELF6-overexpressing cells

Studies have shown that CELF6 overexpression induces G1 phase arrest, while its depletion promotes cell cycle progression, proliferation, and colony formation . This effect appears to be p53 and/or p21 dependent, highlighting the importance of these pathways in CELF6's antiproliferative functions.

How can transcriptomic analyses be integrated with CELF6 antibody-based studies?

Integrating transcriptomic approaches with CELF6 protein analysis provides comprehensive insights into its biological functions:

• RNA-seq after CELF6 modulation:

  • Perform RNA-seq after CELF6 overexpression or knockdown

  • Identify differentially expressed genes (DEGs)

  • Validate selected DEGs by RT-qPCR and protein-level changes by Western blot

  • In A549 lung cancer cells, CELF6 overexpression resulted in 417 up-regulated and 1,351 down-regulated DEGs

• Pathway analysis of CELF6-regulated genes:

  • Perform Gene Ontology (GO) and KEGG pathway analysis

  • Connect transcriptomic changes to cellular phenotypes

  • Down-regulated DEGs after CELF6 overexpression were enriched in immune/inflammation response-related pathways and cell adhesion molecules (CAMs)

• Alternative splicing analysis:

  • Examine exon usage and alternative splicing events after CELF6 modulation

  • Validate splicing changes using RT-PCR with isoform-specific primers

  • Correlate splicing changes with functional outcomes

• Integrative multi-omics approaches:

  • Combine transcriptomic data with ChIP-seq, CLIP-seq, and proteomics

  • Construct regulatory networks to understand CELF6's global impact

  • Identify direct versus indirect regulatory effects

This integrated approach revealed that CELF6 overexpression in lung cancer cells affects genes involved in TNF signaling pathway and cytokine-cytokine receptor interaction, suggesting broader roles in cancer immunity and progression .

How have CELF6 antibodies contributed to understanding autism spectrum disorders?

CELF6 has been implicated in autism spectrum disorders (ASDs) through genetic and functional studies. CELF6 antibodies have facilitated several key discoveries:

• Neural expression mapping:

  • CELF6 antibodies enabled characterization of expression patterns in neuronal populations

  • Identified enrichment in serotonergic neurons, which are implicated in repetitive behaviors and resistance to change that characterize autism

  • Allowed correlation of CELF6 expression with specific neural circuits

• Genetic variant analysis:

  • Immunoblotting was used to validate CELF6 expression in samples with identified genetic variants

  • Analysis of common variants near CELF6 in the Autism Genetic Resource Exchange (AGRE) collection implicated CELF6 in autism risk

  • Screening for rare variants using allele-specific PCR allowed further genetic characterization

• Animal model development and validation:

  • CELF6 antibodies were essential for confirming knockout efficiency in Celf6-/- mouse models

  • These models were generated by deleting exon 4 of Celf6 using Cre-lox recombination

  • Provided tools for studying behavioral phenotypes relevant to autism

• Translational profiling:

  • The bacTRAP methodology allowed cell type-specific analysis of CELF6 in serotonergic neurons

  • Identified several thousand serotonergic-cell expressed transcripts, with 174 highly enriched

  • CELF6 emerged as a promising candidate gene through this approach

These studies highlight CELF6's potential role in neurodevelopmental processes relevant to autism and establish it as a valuable target for further investigation in neuropsychiatric research.

What experimental approaches are recommended for studying CELF6 in neuronal systems?

Investigating CELF6 in neuronal contexts requires specialized experimental approaches:

• Cell type-specific analysis:

  • Use of bacTRAP (translating ribosome affinity purification) to isolate cell type-specific mRNAs

  • Single-cell RNA sequencing combined with CELF6 immunostaining

  • Patch-seq methods to correlate CELF6 expression with electrophysiological properties

• Neural circuit mapping:

  • Immunohistochemistry with CELF6 antibodies combined with neuronal subtype markers

  • Triple labeling approaches to identify specific neuronal populations expressing CELF6

  • Brain region-specific analysis focusing on circuits relevant to behaviors affected in autism

• Functional studies:

  • Celf6 knockout or knockdown in primary neuronal cultures

  • Analysis of dendritic morphology, synapse formation, and neuronal activity

  • Electrophysiological assessment of neurons with altered CELF6 expression

• Behavioral analysis:

  • Comprehensive behavioral testing of Celf6-/- mice

  • Focus on behaviors relevant to autism spectrum disorders

  • Correlation of behavioral phenotypes with molecular and cellular changes

These approaches have successfully identified CELF6's expression in serotonergic neurons and implicated it in autism risk , providing a foundation for deeper investigation of its neurobiological functions.

How can researchers distinguish between CELF6 and other CELF family members in experimental contexts?

Distinguishing CELF6 from other CELF family members is crucial for accurate experimental outcomes:

• Antibody selection strategies:

  • Use antibodies raised against peptides specifically selected for "relative uniqueness across the Celf family"

  • The peptide sequence QPGSDTLYNNGVSPC has been validated for specificity in both Western blot and immunofluorescence applications

  • Always validate commercial antibodies using appropriate controls

• Expression pattern analysis:

  • Compare expression patterns of different CELF family members using specific antibodies

  • RNA-seq or RT-qPCR analysis with isoform-specific primers

  • Consider tissue and developmental stage-specific expression differences

• Functional discrimination:

  • Design rescue experiments with CELF6 versus other CELF proteins

  • Analyze binding specificities and RNA targets

  • Evaluate functional outcomes of overexpressing different family members

• Knockout validation:

  • Use Celf6-/- samples as negative controls for antibody specificity

  • Ensure that antibodies don't cross-react with other CELF proteins in knockout samples

  • Consider compensatory changes in other CELF family members after CELF6 manipulation

Researchers successfully generated CELF6-specific antibodies by selecting peptides based on cross-species conservation, uniqueness across the CELF family, and hydrophobicity, providing a model for specific antibody development .

What are common technical challenges when working with CELF6 antibodies and how can they be resolved?

Researchers frequently encounter technical issues when working with CELF6 antibodies. Here are solutions to common problems:

• Low signal intensity in Western blots:

  • Increase protein loading (30-50 μg recommended)

  • Optimize antibody concentration (try 1:500 instead of 1:1000)

  • Extend primary antibody incubation (overnight at 4°C)

  • Use more sensitive detection methods (e.g., chemiluminescent substrates with extended signal duration)

  • Consider signal amplification systems

• High background in immunofluorescence:

  • Implement more stringent blocking (5% BSA instead of non-fat milk)

  • Increase washing steps (5-6 washes of 10 minutes each)

  • Dilute primary antibody further after titration experiments

  • Pre-absorb antibody with acetone powder from tissues not expressing CELF6

  • Note that only antibodies against specific peptides (e.g., QPGSDTLYNNGVSPC) have been validated for immunofluorescence

• Multiple bands in Western blot:

  • Determine if bands represent isoforms, post-translational modifications, or degradation products

  • Use Celf6-/- samples as negative controls

  • Perform peptide competition assays to identify specific versus non-specific bands

  • Consider using gradient gels for better resolution

• Inconsistent results between experiments:

  • Standardize lysate preparation procedures

  • Establish consistent blocking, antibody incubation, and washing protocols

  • Create detailed standard operating procedures for each application

  • Use internal controls consistently across experiments

Research has shown that not all CELF6 antibodies work equally well across different applications, highlighting the importance of application-specific validation and optimization .

How can researchers optimize immunoprecipitation protocols for studying CELF6-associated complexes?

Immunoprecipitation (IP) of CELF6 requires careful optimization:

• Lysis buffer selection:

  • For RNA-binding proteins like CELF6, use non-denaturing lysis buffers

  • Consider buffers containing low concentrations of NP-40 or Triton X-100

  • Include RNase inhibitors if studying RNA-protein complexes

  • Add protease and phosphatase inhibitors to preserve native protein interactions

• Antibody selection and coupling:

  • Use antibodies validated for IP applications

  • Consider direct coupling to beads (using crosslinkers like BS3 or DMP) to avoid IgG contamination

  • For challenging IPs, compare different antibodies targeting distinct CELF6 epitopes

• Optimization strategies:

  • Adjust antibody-to-lysate ratio

  • Test different incubation times and temperatures

  • Compare various washing stringencies

  • Pre-clear lysates thoroughly before adding CELF6 antibody

• Controls and validation:

  • Include isotype control antibodies

  • Use CELF6 knockout or knockdown samples as negative controls

  • Verify IP efficiency by Western blotting input, unbound, and eluted fractions

  • Confirm specificity of co-immunoprecipitated proteins by reciprocal IP

• Specialized applications:

  • For RNA immunoprecipitation (RIP), include crosslinking steps

  • For chromatin immunoprecipitation (ChIP), optimize crosslinking and sonication conditions

  • For co-IP studies, consider mild detergent conditions to preserve protein-protein interactions

These optimized IP protocols enable investigation of CELF6's interactions with proteins and RNAs, such as its binding to the 3'UTR of p21 mRNA .

What future directions might expand the research applications of CELF6 antibodies?

Emerging technologies and approaches promise to enhance CELF6 antibody applications:

• Advanced imaging techniques:

  • Super-resolution microscopy for detailed subcellular localization studies

  • Multiplexed imaging to simultaneously visualize CELF6 with multiple markers

  • Live-cell imaging with nanobody-based probes for dynamic studies

  • FRET-based approaches to study CELF6 interactions in living cells

• Single-cell applications:

  • Integration with single-cell RNA sequencing

  • Mass cytometry (CyTOF) incorporating CELF6 antibodies

  • Spatial transcriptomics combined with CELF6 immunostaining

• High-throughput screening applications:

  • CELF6 antibody-based readouts for drug screening

  • CRISPR screens with CELF6 antibody detection

  • Automated imaging platforms for phenotypic analysis

• Therapeutic development:

  • Use of CELF6 antibodies for target validation in drug discovery

  • Development of antibody-drug conjugates if CELF6 shows cell-surface expression in specific contexts

  • Companion diagnostic development for stratifying patients

• Clinical applications:

  • Development of standardized immunohistochemistry protocols for diagnostic use

  • Creation of tissue microarrays for large-scale analysis of CELF6 in cancer progression

  • Correlation of CELF6 expression with treatment response

These future directions will expand our understanding of CELF6's biological functions and potential clinical relevance, particularly in cancer biology and neuropsychiatric disorders where CELF6 has already shown significant promise as a research target .