esrp1 Antibody

What is ESRP1 and why is it important in molecular biology research?

ESRP1 (Epithelial Splicing Regulatory Protein 1) is a nuclear-localized RNA splicing factor that regulates the formation of epithelial cell-specific isoforms. In humans, the canonical protein consists of 681 amino acid residues with a molecular mass of 75.6 kDa . ESRP1 is critical in regulating alternative splicing events during epithelial-to-mesenchymal transition (EMT), a process crucial in development and cancer progression . It specifically regulates the expression of FGFR2-IIIb (an epithelial cell-specific isoform of FGFR2) and controls splicing of important transcripts including CD44, CTNND1, and ENAH . ESRP1 is also involved in inner ear development and auditory hair cell differentiation, with mutations linked to hearing loss . Its cell-type specific expression pattern makes it an important marker for epithelial identity in various research contexts.

What are the common applications for ESRP1 antibodies in research?

ESRP1 antibodies are utilized across multiple experimental platforms:

These applications provide complementary approaches to investigate ESRP1 expression, localization, and function in experimental models .

What are the key considerations for validating ESRP1 antibody specificity?

Validating ESRP1 antibody specificity requires several complementary approaches:

• Positive and negative controls: Use cell lines with known ESRP1 expression (e.g., MCF-7, COLO 320) as positive controls and epithelial-to-mesenchymal transitioned cells as negative controls .

• Blocking peptide verification: Compare antibody reactivity in the presence and absence of blocking peptide to confirm specificity, as demonstrated in western blot analysis of rat colon tissue lysate .

• Molecular weight confirmation: Verify detection at the expected molecular weight (~76 kDa for human ESRP1) .

• Cross-reactivity assessment: Confirm that the antibody does not cross-react with other ESRP family members, particularly ESRP2, which shares structural similarities .

• Knockdown/knockout validation: Perform siRNA knockdown or CRISPR knockout of ESRP1 to validate antibody specificity through diminished or absent signal .

• Isoform detection: When studying alternatively spliced variants, confirm whether the antibody recognizes all five known isoforms of ESRP1 .

These validation steps ensure reliable experimental results and prevent misinterpretation of data in ESRP1 research applications.

How should ESRP1 antibodies be optimized for immunohistochemistry in different tissue types?

Optimizing ESRP1 antibodies for immunohistochemistry requires tissue-specific protocol adjustments:

• Antigen retrieval optimization:

  • For epithelial tissues (lung, colon): Use citrate buffer (pH 6.0) with 15-20 minutes heat treatment in an autoclave at 120°C

  • For tissues with dense extracellular matrix: Target retrieval solution (pH 9.0) often yields better results

• Primary antibody incubation:

  • Dilution range: 1:250-1:1000 depending on antibody source and tissue type

  • Incubation time: Overnight at 4°C typically provides optimal signal-to-noise ratio

• Detection system selection:

  • For high-expression contexts: Standard HRP-based detection systems work effectively

  • For low-expression contexts: Amplification systems like ChemMate EnVision kit provide enhanced sensitivity

• Tissue-specific considerations:

  • Human lung tissue: Shows strong epithelial expression

  • Mouse colon tissue: Requires careful blocking to minimize background

  • Human Bowen disease samples: May require lower antibody concentrations (1:500-1:1000)

• Counterstaining adjustments:

  • Brief hematoxylin counterstaining (30-60 seconds) provides optimal nuclear contrast without masking nuclear ESRP1 signals

These optimization strategies ensure specific detection of ESRP1 across different tissue contexts while minimizing background and maximizing signal specificity.

What are the recommended approaches for studying ESRP1-mediated alternative splicing events?

Investigating ESRP1-mediated splicing regulation requires multi-dimensional approaches:

• Splicing target identification and validation:

  • RT-PCR analysis of known ESRP1 targets (FGFR2, CD44, CTNND1, ENAH) using primers spanning alternatively spliced exons

  • Conventional PCR conditions: Initial denaturation (95°C for 2 min), 30 cycles of amplification (95°C for 1 min, 98°C for 20s, 60°C for 30s), and final extension (72°C for 1 min)

  • Quantitative RT-PCR using SYBR Green on ABI 7300 Fast real-time PCR system with GAPDH normalization

• ESRP1 manipulation strategies:

  • siRNA knockdown using validated sequences (e.g., human ESRP1 (262): 5′-GAGAAGGAGUUGAUCCUGCUGUUCU-3′)

  • Transfection using Lipofectamine RNAiMAX with final siRNA concentration of 5 nM

  • For mechanistic studies, pair ESRP1 knockdown with ESRP2 knockdown to address functional redundancy

• RNA-protein interaction analysis:

  • RNA immunoprecipitation to identify direct ESRP1 binding to target transcripts

  • Focus on GU-rich sequence motifs in ISE/ISS-3 cis-regulatory regions of target mRNAs

• Functional assessment in cellular models:

  • Utilize Py2T cell line with Esrp1/2 double knockout as a model system for studying epithelial-mesenchymal transition

  • In vivo zebrafish models can assess functional consequences of ESRP1 variants

• Isoform-specific detection methods:

  • Isoform-specific antibodies for protein-level validation

  • Primers designed to specifically amplify inclusion/exclusion variants of target exons

These methodological approaches provide a comprehensive framework for dissecting ESRP1's role in alternative splicing regulation.

How can researchers distinguish between ESRP1 and ESRP2 in functional studies?

Distinguishing between ESRP1 and ESRP2 functions requires targeted experimental design:

• Antibody selection for individual detection:

  • Use antibodies targeting non-homologous regions: anti-ESRP1 (Sigma-Aldrich HPA023720) and anti-ESRP2 (Abcam ab113486)

  • Validate specificity through western blot analysis of cells with differential expression

• Selective knockdown/knockout approaches:

  • Individual siRNA targeting with validated sequences:

    • ESRP1: 5′-GAGAAGGAGUUGAUCCUGCUGUUCU-3′ or 5′-CGGAGAAGCUCUGGUUAGGUUUGUA-3′

    • ESRP2: 5′-CCGAGGUGAUAAAGCAGAAAUACGA-3′ or 5′-GCGUCCGCUAUAUUGAGGUGUAUAA-3′

  • Sequential knockdown experiments to identify unique versus redundant functions

• Distinct functional roles assessment:

  • ESRP1 specifically suppresses expression of the Rac1b isoform

  • ESRP2 primarily represses epithelial-mesenchymal transition-inducing transcription factors

  • Evaluate cell motility changes as a functional readout for differential effects

• Expression pattern analysis:

  • Compare expression levels in epithelial tissues using qRT-PCR with specific primers:

    • ESRP1: 5′-CAATATTGCCAAGGGAGGTG-3′ (sense), 5′-GTCCCCATGTGATGTTTGTG-3′ (antisense)

    • ESRP2: 5′-TGCCACAGAGGATGACTTTG-3′ (sense), 5′-ATTGACTGCTGGGCTCTTTG-3′ (antisense)

• Rescue experiments:

  • Perform selective rescue experiments in double knockdown/knockout models

  • Reintroduce either ESRP1 or ESRP2 to identify unique functional contributions

These approaches enable researchers to delineate the specific contributions of ESRP1 versus ESRP2 in RNA splicing regulation and cellular processes.

What are common challenges when detecting ESRP1 in western blot experiments?

When working with ESRP1 in western blot applications, researchers should address these common challenges:

• Multiple band detection issues:

  • Expected molecular weight: 76 kDa for canonical ESRP1

  • Additional bands may represent:

    • Known isoforms (up to 5 reported variants)

    • Post-translational modifications

    • Degradation products (particularly when using proteolysis-sensitive samples)

  • Validation approach: Use positive control lysates from epithelial cell lines (MCF-7, COLO 320)

• Sample preparation considerations:

  • Nuclear protein extraction: Use specialized buffers containing 20 mM Tris-HCl (pH 7.4), 5 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, with protease inhibitors (1% aprotinin, 1 mM PMSF)

  • Sample degradation: Maintain samples on ice and include phosphatase inhibitors for phosphorylation studies

  • Protein measurement: Use BCA protein assay for accurate quantification

• Antibody selection and optimization:

  • Dilution optimization: Test range from 1:500-1:2000 for optimal signal-to-noise ratio

  • Incubation conditions: Overnight at 4°C typically yields best results

  • Secondary antibody selection: HRP-conjugated mouse or rabbit IgG depending on primary antibody host

• Detection sensitivity issues:

  • For low expression samples: Use enhanced chemiluminescent substrates and longer exposure times

  • Background reduction: More thorough blocking (5% skim milk in TBST) and additional washing steps

  • Signal amplification: Consider biotin-streptavidin systems for weak signals

• Quantification considerations:

  • Use appropriate loading controls (α-tubulin) for normalization

  • Analyze band intensity with Image Reader LAS-4000 or similar software for accurate quantification

Addressing these challenges ensures reliable and reproducible western blot results when working with ESRP1.

How should researchers interpret conflicting ESRP1 immunostaining patterns across different tissues?

Interpreting variable ESRP1 immunostaining patterns requires careful consideration of biological and technical factors:

• Biological variability considerations:

  • Cell-type specific expression: ESRP1 shows epithelial cell-specific expression patterns

  • Developmental stage differences: Expression patterns may vary during embryonic development versus adult tissues

  • Disease state influence: Upregulated during carcinogenesis but downregulated at invasive fronts

  • EMT status: Expression decreases during epithelial-mesenchymal transition

• Technical variation factors:

  • Antibody epitope accessibility: Different antibodies target distinct regions of ESRP1 (N-terminal vs. C-terminal)

  • Fixation effects: Formalin fixation can mask epitopes differently across tissue types

  • Antigen retrieval differences: pH 6.0 citrate buffer versus pH 9.0 target retrieval solution efficacy varies by tissue

  • Detection system sensitivity: Chromogenic versus fluorescent detection systems may reveal different expression patterns

• Validation approach for conflicting results:

  • Multi-antibody validation: Use at least two antibodies targeting different epitopes:

    • N-terminal antibodies (amino acids 30-80)

    • C-terminal antibodies (amino acids 600 to C-terminus)

  • Complementary methods: Validate with RNA in situ hybridization or RT-PCR

  • Functional validation: Correlate with known ESRP1-regulated splicing events in the tissue

• Context-specific interpretation framework:

  • Cancer tissues: Consider heterogeneity and EMT status at tumor margins

  • Developmental contexts: Interpret based on known temporal expression patterns

  • Pathological samples: Compare with matched normal tissues

This interpretative framework helps researchers reconcile apparently conflicting immunostaining patterns and extract meaningful biological insights.

What considerations are important when analyzing ESRP1 antibody immunoreactivity in cancer studies?

Cancer-specific ESRP1 analysis requires nuanced interpretation strategies:

• Expression pattern considerations:

  • Tumor heterogeneity: ESRP1 expression decreases at invasive fronts but may be upregulated in tumor cores

  • EMT status markers: Co-stain with E-cadherin to correlate ESRP1 expression with epithelial phenotype

  • Spatial distribution: Assess nuclear localization intensity and distribution patterns across tumor regions

• Prognostic/diagnostic value assessment:

  • Compare expression between tumor subtypes

  • Correlate with clinical parameters and outcomes

  • Analyze relationship with EMT-inducing transcription factors (δEF1, SIP1)

• Functional correlation strategies:

  • Assess Rac1b isoform expression as a downstream ESRP1 target

  • Examine cell motility markers in relation to ESRP1 expression

  • Analyze alternative splicing patterns of FGFR2, CD44, and CTNND1

• Technical optimization for cancer tissues:

  • Antigen retrieval: More aggressive retrieval often needed for heavily fixed tumor tissues

  • Background reduction: Additional blocking steps to minimize non-specific binding

  • Multi-antibody approach: Use both antibodies recognizing different epitopes for validation

• Controls and validation framework:

  • Internal controls: Use adjacent normal tissue when available

  • Positive controls: Include known ESRP1-expressing epithelial cells

  • Negative controls: EMT regions with expected low expression

  • Functional validation: siRNA approaches in derived cell lines

These considerations support accurate interpretation of ESRP1 expression patterns in cancer contexts and strengthen translational research applications.

How can ESRP1 antibodies be utilized in research on epithelial-mesenchymal transition (EMT)?

ESRP1 antibodies provide powerful tools for investigating EMT processes:

• Temporal profiling during EMT progression:

  • Immunostaining time-course experiments with ESRP1 antibodies (1:100 dilution) during induced EMT

  • Co-staining with E-cadherin (1:100, BD Biosciences 610181) to correlate epithelial marker loss with ESRP1 downregulation

  • Western blot analysis at multiple timepoints to quantify ESRP1 protein level changes

• Mechanistic studies on splicing regulation during EMT:

  • Immunoprecipitation of ESRP1-containing complexes to identify protein interactions changing during EMT

  • RNA-IP to map shifts in ESRP1-bound transcripts across EMT progression

  • Correlation with alternative splicing patterns of key EMT-related transcripts:

    • FGFR2 (IIIb to IIIc switch)

    • CD44 (variable exon inclusion)

    • CTNND1 (p120-catenin isoform switching)

• Reversibility assessment in mesenchymal-epithelial transition (MET):

  • Track ESRP1 re-expression during induced MET

  • Correlate with reversion of EMT-associated splicing events

  • Use antibodies against both ESRP1 and ESRP2 to assess their relative contributions

• Therapeutic intervention studies:

  • Monitor ESRP1 levels as readout for EMT-targeting therapies

  • Assess splicing pattern normalization following treatment

  • Correlate with invasive/migratory phenotype changes

• Clinical correlation approach:

  • Immunohistochemical analysis of tumor sections focusing on:

    • Invasive fronts (expected ESRP1 downregulation)

    • Tumor core (variable ESRP1 expression)

    • Metastatic sites (potential ESRP1 re-expression during MET)

These applications leverage ESRP1 antibodies as powerful tools for dissecting the molecular mechanisms underlying EMT/MET processes in development and disease.

What approaches can be used to study ESRP1 gene variants and their functional consequences?

Investigating ESRP1 gene variants requires multi-dimensional experimental approaches:

• Variant identification and prioritization strategies:

  • Source variants from repositories like Gabriella Miller Kids First Program and ClinVar

  • Focus on variants associated with specific phenotypes (orofacial cleft, hearing loss)

  • Prioritize variants in functional domains (RNA recognition motifs) or affecting conserved residues

• In silico prediction approaches:

  • Apply multiple predictive tools while recognizing their limitations

  • Weight experimental validation higher than in silico predictions per ACMG-AMP guidelines

  • Focus on variants with consistent predictions across multiple algorithms

• Functional validation in cellular models:

  • Clone variants using site-directed mutagenesis

  • Express variants in ESRP1-knockout cell lines

  • Assess splicing regulation of known ESRP1 targets:

    • RT-PCR analysis of FGFR2, CD44, CTNND1 splicing patterns

    • Western blot analysis of resulting protein isoforms

• In vivo functional testing:

  • Zebrafish models: Inject variant ESRP1 mRNA into esrp1-/-; esrp2-morpholino embryos

  • Assess rescue of cleft anterior neurocranium phenotype

  • Compare variant function to wild-type in developmental contexts

• Biochemical characterization:

  • RNA binding assays to assess variant effects on GU-rich motif recognition

  • Protein stability and subcellular localization analysis

  • Protein-protein interaction studies to identify altered molecular partnerships

• Disease-specific functional assays:

  • For hearing loss variants: Focus on inner ear development markers

  • For orofacial cleft variants: Examine periderm development indicators

  • For cancer-related variants: Assess EMT regulation capacity

These approaches enable comprehensive characterization of ESRP1 variants and their potential contribution to human disease phenotypes.

How can researchers investigate ESRP1 and ESRP2 redundancy versus distinct functions?

Dissecting redundant versus unique functions of ESRP1/ESRP2 requires strategic experimental designs:

• Expression pattern comparative analysis:

  • Quantitative RT-PCR with gene-specific primers :

    • ESRP1: 5′-CAATATTGCCAAGGGAGGTG-3′ (sense), 5′-GTCCCCATGTGATGTTTGTG-3′ (antisense)

    • ESRP2: 5′-TGCCACAGAGGATGACTTTG-3′ (sense), 5′-ATTGACTGCTGGGCTCTTTG-3′ (antisense)

  • Immunostaining with specific antibodies:

    • Anti-ESRP1 (Sigma-Aldrich HPA023720)

    • Anti-ESRP2 (Abcam ab113486)

  • RNA-seq analysis across tissue types and developmental stages

• Single versus double knockdown/knockout experiments:

  • Individual siRNA knockdown :

    • ESRP1-specific: 5′-GAGAAGGAGUUGAUCCUGCUGUUCU-3′

    • ESRP2-specific: 5′-CCGAGGUGAUAAAGCAGAAAUACGA-3′

  • Sequential knockdown to assess compensatory upregulation

  • Double knockout cell models (e.g., Esrp1/2 double knockout Py2T cells)

• Target-specific functional analysis:

  • Compare splicing patterns after individual versus combined knockdown

  • Identify ESRP1-unique targets: Focus on Rac1b isoform expression

  • Identify ESRP2-unique targets: Examine EMT-inducing transcription factors

• Rescue experiments with selective complementation:

  • Express ESRP1 in double knockout background to identify ESRP1-specific functions

  • Express ESRP2 in double knockout background to identify ESRP2-specific functions

  • Create chimeric proteins to map domain-specific functions

• Model organism comparative studies:

  • Zebrafish single versus double mutant phenotypes

  • Tissue-specific conditional knockout models

  • Developmental stage-specific gene inactivation

• Biochemical property comparison:

  • RNA binding specificity assessment

  • Protein interaction network mapping

  • Post-translational modification patterns

These approaches provide a comprehensive framework for delineating shared versus distinct functions of ESRP proteins in development and disease contexts.

How can ESRP1 antibodies be utilized in single-cell research applications?

Adapting ESRP1 antibody applications for single-cell analysis requires specialized approaches:

• Single-cell immunofluorescence optimization:

  • Signal amplification strategies for low-abundance detection

  • Multiplex staining with lineage markers and EMT indicators

  • Quantitative image analysis workflows for single-cell expression measurement

  • Z-stack acquisition for accurate subcellular localization

• Flow cytometry applications:

  • Intracellular staining protocols with fixation and permeabilization optimization

  • Antibody titration to determine optimal concentration for flow applications

  • Gating strategies to identify ESRP1-high versus ESRP1-low populations

  • Index sorting for correlation with single-cell transcriptomics

• Single-cell proteomics integration:

  • Application in CyTOF/mass cytometry panels

  • Correlation with epithelial-mesenchymal spectrum markers

  • Computational analysis to identify cell state transitions

  • Trajectory mapping during developmental or disease processes

• Spatial single-cell applications:

  • Optimization for highly multiplexed imaging platforms

  • Co-detection with RNA by combined IF/RNA-FISH approaches

  • Spatial mapping of ESRP1 expression in tissue contexts

  • Neighborhood analysis to identify cellular interactions affecting ESRP1 expression

• Single-cell multi-omics integration:

  • CITE-seq approaches to correlate protein and transcript levels

  • Integration with single-cell RNA splicing analysis

  • Correlation with chromatin accessibility at single-cell resolution

These emerging applications position ESRP1 antibodies as valuable tools for dissecting cellular heterogeneity in epithelial tissues and during state transitions at unprecedented resolution.

What are considerations for using ESRP1 antibodies in high-throughput screening applications?

Implementing ESRP1 antibodies in high-throughput screening requires systematic optimization:

• Assay miniaturization and automation:

  • Microplate format optimization (96/384/1536-well)

  • Automated liquid handling compatibility assessment

  • Signal detection optimization for plate reader systems

  • Z-factor determination for assay quality control

• Readout system selection and validation:

  • Fluorescence intensity-based detection systems

  • High-content imaging platforms for subcellular localization

  • Homogeneous assay formats to minimize washing steps

  • Dual-reporter systems for normalization

• Screening library considerations:

  • Compound libraries targeting splicing machinery

  • siRNA/CRISPR libraries for genetic modifier screens

  • cDNA expression libraries to identify regulators

  • Targeted libraries based on known ESRP1 interactors

• Control development and validation:

  • Positive controls: EMT inducers (TGFβ) for ESRP1 downregulation

  • Negative controls: MET inducers for ESRP1 upregulation

  • Internal controls: Housekeeping proteins for normalization

  • Dynamic range assessment across control conditions

• Data analysis and hit selection strategies:

  • Normalization methods for plate and positional effects

  • Statistical approaches for hit identification

  • Secondary assay cascades for hit validation

  • Orthogonal approaches to confirm mechanism of action

• Scale-up considerations:

  • Antibody batch consistency across screening campaign

  • Day-to-day variability assessment

  • Edge effects mitigation strategies

  • Automation compatibility validation

These considerations enable successful implementation of ESRP1 antibodies in high-throughput screening campaigns to identify modulators of epithelial splicing programs.

How can researchers effectively combine ESRP1 antibody approaches with RNA-sequencing for comprehensive splicing analysis?

Integrating protein-level and RNA-level analyses provides complementary insights into ESRP1 function:

• Experimental design strategies:

  • Paired samples for both antibody-based detection and RNA-seq analysis

  • Time-course designs to capture dynamic changes

  • Inclusion of appropriate controls:

    • ESRP1 knockdown/knockout

    • ESRP1 overexpression

    • EMT induction models

• RNA-seq approach optimization:

  • Library preparation methods optimized for alternative splicing detection

  • Sequencing depth recommendations: minimum 50-60 million paired-end reads

  • Read length considerations: 100-150bp paired-end reads for improved junction detection

  • Strand-specific protocols for accurate isoform discrimination

• Antibody-based validation approaches:

  • Western blot confirmation of ESRP1 protein levels

  • Immunofluorescence to confirm cellular distribution

  • Co-IP to identify interacting partners affecting splicing

  • ChIP-seq to identify potential transcriptional regulatory roles

• Integrative bioinformatics workflows:

  • Splicing event quantification tools (rMATS, SUPPA2, Whippet)

  • Motif analysis near differentially spliced regions (ESRP1 binding motifs)

  • Correlation between ESRP1 protein levels and splicing pattern changes

  • Pathway analysis of differentially spliced transcripts

• Validation of key splicing events:

  • RT-PCR confirmation of identified splicing changes

  • Minigene reporter assays for mechanistic validation

  • Protein isoform detection by western blot

  • Functional studies of alternatively spliced protein variants

• Advanced integrative approaches:

  • RNA-IP-seq to directly map ESRP1-bound transcripts

  • eCLIP to identify precise binding sites at nucleotide resolution

  • Ribosome profiling to assess translational impact of splicing changes

  • Proteomics to confirm protein isoform expression