igf2bp3 Antibody

What is IGF2BP3 and why is it significant in research?

IGF2BP3 (insulin-like growth factor 2 mRNA binding protein 3) is an RNA-binding protein that plays a critical role in regulating mRNA stability and translation, which is essential for cellular growth and differentiation . It is predominantly localized in the cytoplasm, where it binds to specific mRNAs including IGF-II and c-Myc, influencing their stability and translation efficiency . Its significance in research stems from its high expression in embryonic tissues and various cancers while showing minimal expression in normal adult tissues, making it a potential biomarker for cancer diagnosis and prognosis . IGF2BP3 has been particularly implicated in lung cancer development, where it promotes tumorigenesis by attenuating p53 protein stability .

What are the primary applications for IGF2BP3 antibodies in cancer research?

IGF2BP3 antibodies are employed in multiple applications critical for cancer research:

• Western Blotting (WB): For detecting and quantifying IGF2BP3 protein levels in cell lysates and tissue samples

• Immunoprecipitation (IP): To isolate IGF2BP3 and its binding partners for protein-protein interaction studies

• Immunofluorescence (IF): For visualizing subcellular localization of IGF2BP3 in cancer cells

• Immunohistochemistry with paraffin-embedded sections (IHCP): For examining IGF2BP3 expression in patient tumor samples

• Flow Cytometry (FCM): For quantifying IGF2BP3 expression at the cellular level

• ELISA: For quantitative measurement of IGF2BP3 levels in biological samples

These techniques have revealed that IGF2BP3 is expressed in 27–55% of primary pulmonary adenocarcinoma cases and 75–90% of squamous cell carcinoma cases of the lung, with high expression correlating with poor prognosis .

What types of IGF2BP3 antibodies are available and how should I select one for my experiment?

Several types of IGF2BP3 antibodies are available for research purposes:

When selecting an antibody, consider:

• Target species reactivity (human, mouse, rat)

• Required applications (western blot, immunohistochemistry, etc.)

• Validated specificity (check literature citations and validation data)

• Host species compatibility with your experimental system

• Whether conjugated forms are needed for your detection method

For detecting IGF2BP3 in human samples, the mouse monoclonal IGF2BP3 antibody (C-11) has been extensively validated for multiple applications including western blotting, immunoprecipitation, and immunohistochemistry .

How can I optimize western blotting protocols for IGF2BP3 detection in different tissue types?

Optimizing western blotting for IGF2BP3 detection requires careful consideration of tissue-specific variables:

• Sample preparation:

  • For cancer tissues: Use RIPA buffer supplemented with protease inhibitors to extract total protein while preserving IGF2BP3

  • For cell lines: HeLa, A549, HEK-293, HepG2, Jurkat, K-562, HSC-T6, and NIH/3T3 cells have all been validated for IGF2BP3 detection

• Protein loading and separation:

  • Load 20-50 μg of total protein per lane

  • Use 8-10% SDS-PAGE gels for optimal resolution of the 64 kDa IGF2BP3 protein

• Antibody dilution:

  • Primary antibody: Titrate within 1:5000-1:50000 range depending on expression levels

  • The specific dilution may need to be optimized for each tissue type

• Detection considerations:

  • IGF2BP3 is observed at approximately 64 kDa

  • Use appropriate positive controls (A549 lung cancer cells show high expression levels)

  • Include negative controls (normal lung tissues typically show negligible expression)

• Troubleshooting tissue-specific issues:

  • For tissues with high lipid content: Additional washing steps may be required

  • For tissues with low IGF2BP3 expression: Consider immunoprecipitation before western blotting to concentrate the protein

When comparing expression across different tissue types, normalize to appropriate housekeeping proteins and consider using increased antibody concentration for tissues with naturally lower IGF2BP3 expression .

What are the critical factors for successful immunohistochemical detection of IGF2BP3 in tumor samples?

Successful immunohistochemical detection of IGF2BP3 in tumor samples depends on several critical factors:

• Tissue fixation and processing:

  • Formalin-fixed, paraffin-embedded sections are most commonly used

  • Optimal fixation time (24 hours) is crucial to preserve epitope accessibility

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Optimization of retrieval time is essential for maximum sensitivity

• Blocking and antibody incubation:

  • Thorough blocking of endogenous peroxidase activity

  • Use of protein blockers to reduce background staining

  • Optimal primary antibody dilution must be determined empirically

  • Longer incubation times (overnight at 4°C) may improve sensitivity

• Signal interpretation:

  • IGF2BP3 staining is observed in both nucleus and cytoplasm in cancer tissues

  • Positive staining in normal tissues is rare, making it a good negative control

  • Squamous cell carcinoma typically shows higher expression (26/44 cases) compared to adenocarcinoma (4/15 cases)

• Scoring systems:

  • Consider intensity (weak, moderate, strong) and percentage of positive cells

  • IGF2BP3 positivity correlates with high-grade lung cancers (p = 0.047)

For research comparing different tumor grades or types, maintaining consistent staining conditions across all samples is crucial for valid comparisons of IGF2BP3 expression patterns .

How can I effectively co-immunoprecipitate IGF2BP3 with its interaction partners?

Effectively co-immunoprecipitating IGF2BP3 with its binding partners requires careful optimization:

• Lysis buffer selection:

  • For protein-protein interactions: Use mild NP-40 or CHAPS-based buffers

  • For RNA-binding studies: Use RNA-preserving buffers with RNase inhibitors

  • When studying USP10 interaction: Incorporate deubiquitinase inhibitors to preserve interactions

• Antibody selection and immobilization:

  • Use monoclonal IGF2BP3 antibodies for higher specificity in pull-downs

  • Pre-clearing lysates with appropriate control IgG is essential

  • Consider crosslinking antibodies to beads to prevent antibody contamination in eluates

• Protocol optimization for specific partners:

  • For USP10 interaction: Use low-salt washing conditions to preserve their association

  • For p53-related complexes: Consider crosslinking approaches to capture transient interactions

  • For RNA-binding partners: Treatment with RNase can help distinguish direct protein-protein interactions from RNA-mediated associations

• Elution and detection strategies:

  • Gentle elution with antibody-specific peptides can help maintain complex integrity

  • Western blot analysis should include verification of both IGF2BP3 and suspected binding partners

  • Mass spectrometry analysis of co-immunoprecipitated complexes can identify novel interaction partners

The research revealing IGF2BP3's direct association with deubiquitinase USP10 demonstrates the value of this approach, as this interaction was shown to attenuate USP10's function in stabilizing p53 protein . This finding provided critical insights into IGF2BP3's role in lung tumorigenesis through p53 regulation.

How should I design experiments to investigate IGF2BP3's role in cancer cell proliferation and invasion?

Designing robust experiments to investigate IGF2BP3's role in cancer requires multi-level approaches:

• Expression manipulation strategies:

  • Overexpression: Use lentiviral/plasmid vectors with full-length IGF2BP3 cDNA

  • Knockdown: Apply siRNA or shRNA targeting conserved regions of IGF2BP3

  • CRISPR/Cas9: For complete knockout studies in cell lines

  • Use A549 cells (high IGF2BP3 expression) and H460 cells (low expression) as experimental models

• Proliferation assays:

  • MTT or CCK-8 assays for measuring metabolic activity

  • BrdU incorporation for assessing DNA synthesis

  • Colony formation for long-term growth effects

  • Cell cycle analysis by flow cytometry to assess G0/G1 arrest

• Migration and invasion assays:

  • Wound healing assays to assess cell migration

  • Transwell or Boyden chamber assays with or without Matrigel coating

  • 3D spheroid invasion assays for more physiologically relevant models

• In vivo models:

  • Subcutaneous xenograft models to assess tumor growth

  • Orthotopic models for tissue-specific effects

  • Tail vein injection for metastasis assessment

• Molecular mechanism investigations:

  • Assess p53 protein stability through cycloheximide chase assays

  • Measure half-life of p53 after IGF2BP3 silencing

  • Examine USP10-p53 axis through co-immunoprecipitation

When interpreting results, it's important to compare the effects of IGF2BP3 manipulation across multiple cell lines and validate findings using both in vitro and in vivo models, as demonstrated in studies showing that IGF2BP3 overexpression promotes cell proliferation, tumor migration, and invasion both in vitro and in vivo .

What approaches can I use to study the interaction between IGF2BP3 and p53 stability pathways?

To comprehensively study IGF2BP3's influence on p53 stability pathways:

• Protein stability assessment:

  • Cycloheximide chase assays: Treat cells with cycloheximide to block new protein synthesis and monitor p53 degradation rates with and without IGF2BP3 manipulation

  • Pulse-chase labeling: Use radioactive amino acids to label newly synthesized proteins and track p53 half-life

  • Ubiquitination assays: Immunoprecipitate p53 and probe for ubiquitin to assess ubiquitination levels

• USP10-IGF2BP3-p53 axis investigation:

  • Reciprocal co-immunoprecipitation to confirm interactions between IGF2BP3, USP10, and p53

  • In vitro deubiquitination assays with purified proteins to directly test IGF2BP3's effect on USP10 activity

  • Domain mapping experiments to identify critical regions of IGF2BP3 responsible for USP10 interaction

• Cellular localization studies:

  • Immunofluorescence co-localization of IGF2BP3, USP10, and p53

  • Cell fractionation to determine compartment-specific interactions

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

• Functional readouts:

  • p53 target gene expression (p21, MDM2, BAX) by qRT-PCR and western blotting

  • Apoptosis assays following DNA damage in IGF2BP3-manipulated cells

  • Cell cycle analysis to confirm G0/G1 arrest effects

• Rescue experiments:

  • Concurrent manipulation of IGF2BP3 and USP10 to determine epistatic relationships

  • Introduction of non-degradable p53 mutants to bypass IGF2BP3 effects

  • Expression of IGF2BP3 mutants lacking USP10 binding capacity

Research has demonstrated that silencing IGF2BP3 expression in lung cancer cells increases both the half-life and protein level of p53, inducing G0/G1 arrest, which supports IGF2BP3's role in promoting lung tumorigenesis through attenuating p53 protein stability .

How can I differentiate between IGF2BP3's RNA-binding functions and its protein interaction effects in cancer progression?

Differentiating between IGF2BP3's RNA-binding and protein interaction functions requires strategic experimental approaches:

• Domain-specific mutant analysis:

  • Generate IGF2BP3 constructs with mutations in RNA-recognition motifs (RRMs) that abolish RNA binding

  • Create IGF2BP3 mutants defective in protein-protein interaction domains while maintaining RNA binding

  • Compare phenotypic effects of these mutants in functional assays

• RNA-protein interaction studies:

  • RNA immunoprecipitation (RIP) to identify bound RNA targets

  • Cross-linking immunoprecipitation (CLIP) for higher resolution mapping of RNA binding sites

  • RNA electrophoretic mobility shift assays (EMSA) to assess direct RNA binding capabilities

  • RNA-seq analysis following IGF2BP3 manipulation to identify globally affected transcripts

• Protein interaction network analysis:

  • Immunoprecipitation followed by mass spectrometry to identify protein binding partners

  • Yeast two-hybrid screening for direct protein interactors

  • Proximity-dependent biotin identification (BioID) to identify proteins in the same complex

  • Compare interactome with and without RNase treatment to distinguish RNA-dependent interactions

• Functional separation strategies:

  • RNase treatment in cell lysates before co-immunoprecipitation to eliminate RNA-dependent interactions

  • Use of RNA-binding deficient mutants in USP10/p53 interaction studies

  • Analysis of target mRNA stability versus protein stability effects

• Integrated analysis:

  • Correlate changes in target mRNA levels with changes in corresponding protein levels

  • Assess whether IGF2BP3's effects on p53 protein stability occur independently of changes in p53 mRNA levels

  • Determine if USP10 interaction is dependent on IGF2BP3's RNA-binding capacity

Research has revealed that IGF2BP3 directly associates with the deubiquitinase USP10 and attenuates its function in stabilizing p53 protein, indicating a protein interaction effect that appears distinct from its canonical RNA-binding functions in promoting lung tumorigenesis .

How reliable is IGF2BP3 immunohistochemistry for distinguishing between different cancer subtypes?

The reliability of IGF2BP3 immunohistochemistry for cancer subtype differentiation varies by tissue type and has specific strengths and limitations:

• Lung cancer subtyping:

  • IGF2BP3 shows differential expression patterns: positive in 75-90% of squamous cell carcinoma cases versus 27-55% of adenocarcinoma cases

  • Tissue microarray studies confirm higher expression in squamous cell carcinoma (26/44 cases, 59.1%) compared to adenocarcinoma (4/15 cases, 26.7%)

  • The differential expression pattern makes it potentially useful for subtyping lung cancers

• Tumor grade differentiation:

  • Statistical analysis shows significant correlation between IGF2BP3 expression and high-grade lung cancers (p = 0.047)

  • IGF2BP3 serves as a marker for carcinomas and high-grade dysplastic lesions of pancreatic ductal epithelium

  • This correlation with tumor grade enhances its utility in prognostic assessment

• Diagnostic sensitivity and specificity:

  • Negligible expression in normal adult tissues provides excellent specificity

  • Variable sensitivity across different cancer types limits universal application

  • Combined use with other markers may enhance diagnostic accuracy

• Technical considerations affecting reliability:

  • Standardized staining protocols are essential for consistent results

  • Scoring systems must be consistent across studies for comparable data

  • Both nuclear and cytoplasmic staining should be evaluated

• Validation recommendations:

  • Parallel testing with established markers for specific cancer subtypes

  • Correlation with molecular testing (e.g., RNA expression analysis)

  • Inclusion of appropriate controls in each staining batch

While IGF2BP3 immunohistochemistry shows promise for distinguishing between cancer subtypes, particularly in lung cancer, its optimal use may be as part of a panel of markers rather than as a standalone diagnostic tool .

What considerations are important when using IGF2BP3 as a prognostic biomarker in cancer research?

When employing IGF2BP3 as a prognostic biomarker in cancer research, several key considerations must be addressed:

While IGF2BP3 shows promise as a prognostic biomarker, particularly in lung cancer where its high expression predicts poor prognosis, robust validation studies with standardized methodologies are necessary to establish its clinical utility .

How can I validate the specificity of IGF2BP3 antibodies for immunohistochemical applications in clinical samples?

Validating IGF2BP3 antibody specificity for clinical immunohistochemistry requires systematic multi-step approaches:

• Positive and negative tissue controls:

  • Positive controls: Fetal liver, lung, kidney, or placenta tissues which naturally express IGF2BP3

  • Negative controls: Normal adult tissues which typically lack IGF2BP3 expression

  • Cancer tissue with known IGF2BP3 expression (e.g., squamous cell carcinoma of lung)

• Antibody validation experiments:

  • Western blot confirmation using positive cell lines (A549, HeLa, HEK-293)

  • Peptide competition assays to demonstrate binding specificity

  • Comparison of staining patterns across multiple antibody clones targeting different epitopes

  • Correlation with mRNA expression by in situ hybridization or RT-PCR

• Technical controls during IHC protocol:

  • Isotype control antibodies to assess non-specific binding

  • Primary antibody omission controls

  • Absorption controls using immunizing peptide

  • Dilution series to determine optimal antibody concentration

• Cross-validation approaches:

  • Comparison with alternate detection methods (RNA-scope, RT-PCR)

  • Parallel staining with multiple validated antibodies

  • Correlation with genetic knockdown/knockout controls in cell lines

  • Digital image analysis for quantitative assessment of staining patterns

• Documentation for clinical validation:

  • Record batch-to-batch consistency of antibody performance

  • Document reproducibility across different operators and laboratories

  • Assess stability of staining over time in archived samples

  • Maintain detailed protocols for future reference and standardization

When validating IGF2BP3 antibodies, it's particularly important to confirm the dual nuclear and cytoplasmic staining pattern observed in cancer tissues, and to verify the absence of staining in normal adult tissues, which serves as a critical negative control for specificity assessment .

What experimental approaches can investigate IGF2BP3's role in modulating the tumor microenvironment?

Investigating IGF2BP3's influence on the tumor microenvironment requires sophisticated experimental approaches:

• Co-culture systems:

  • Cancer cells with manipulated IGF2BP3 expression co-cultured with fibroblasts, immune cells, or endothelial cells

  • Transwell systems to study paracrine effects without direct cell contact

  • 3D organoid models incorporating multiple cell types for physiologically relevant interactions

• Secretome analysis:

  • Conditioned media profiling from IGF2BP3-manipulated cancer cells using mass spectrometry

  • Cytokine/chemokine arrays to identify altered secretion patterns

  • Exosome isolation and characterization to study IGF2BP3's influence on intercellular communication

• Immune interaction studies:

  • Flow cytometric analysis of tumor-infiltrating immune cells in IGF2BP3-high versus IGF2BP3-low tumors

  • Assessment of PD-L1 expression in relation to IGF2BP3 levels

  • Functional T-cell activation assays in the presence of IGF2BP3-manipulated cancer cells

• Angiogenesis assessment:

  • Tube formation assays using endothelial cells exposed to conditioned media

  • Analysis of angiogenic factors (VEGF, bFGF) expression in relation to IGF2BP3 status

  • In vivo imaging of tumor vasculature in IGF2BP3-manipulated xenografts

• Extracellular matrix remodeling:

  • Analysis of matrix metalloproteinase expression and activity

  • Collagen contraction assays to assess cancer-associated fibroblast activation

  • Second harmonic generation microscopy to visualize ECM structure alterations

• In vivo approaches:

  • Humanized mouse models with reconstituted immune systems

  • Intravital microscopy to visualize tumor-stroma interactions in real-time

  • Single-cell RNA sequencing of tumor and microenvironment components from IGF2BP3-high versus IGF2BP3-low tumors

While direct research on IGF2BP3's role in modulating the tumor microenvironment is still emerging, its established functions in promoting cancer cell proliferation, migration, and invasion suggest significant potential for influencing tumor-microenvironment interactions that warrant further investigation .

How does IGF2BP3 interact with other RNA-binding proteins in regulating cancer-related mRNAs?

Understanding IGF2BP3's cooperative or competitive interactions with other RNA-binding proteins requires multi-faceted approaches:

• Protein complex identification:

  • RNA-binding protein immunoprecipitation followed by mass spectrometry

  • Proximity labeling approaches (BioID, APEX) to identify proteins in close proximity to IGF2BP3

  • Size exclusion chromatography to isolate and characterize native IGF2BP3-containing complexes

  • Yeast two-hybrid or mammalian two-hybrid screens focused on RNA-binding protein libraries

• Binding site overlap analysis:

  • Cross-linking immunoprecipitation sequencing (CLIP-seq) for IGF2BP3 and other RBPs

  • Bioinformatic analysis to identify shared or exclusive binding motifs and sites

  • Competitive binding assays using purified proteins and synthetic RNA substrates

  • Systematic analysis of binding affinities and kinetics for shared RNA targets

• Functional cooperation or antagonism:

  • Co-depletion or co-overexpression of IGF2BP3 with other RBPs

  • Analysis of target mRNA stability and translation efficiency

  • Ribosome profiling to assess translational impact of combinatorial RBP manipulation

  • CRISPR screens to identify synthetic lethal interactions with IGF2BP3

• Structural biology approaches:

  • Cryo-EM or X-ray crystallography of IGF2BP3-containing RNP complexes

  • NMR studies of domain interactions between IGF2BP3 and partner RBPs

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

• Post-translational modification analysis:

  • Identification of modifications that regulate IGF2BP3's interactions with other RBPs

  • Phosphoproteomics to map signaling-dependent changes in interaction networks

  • Mutational analysis of modification sites to determine functional consequences

While direct evidence of IGF2BP3's interactions with specific RNA-binding proteins in cancer contexts is still emerging, its established role in regulating cancer-related mRNAs suggests potential for both cooperative and competitive interactions with other RBPs that influence cancer progression, especially considering its diverse functions in mRNA stability and translation that likely involve multiple protein partners .

What therapeutic strategies could target IGF2BP3 or its downstream pathways in cancer treatment?

Developing therapeutic strategies targeting IGF2BP3 requires exploration of multiple intervention points:

• Direct targeting approaches:

  • Small molecule inhibitors of IGF2BP3-RNA interactions

  • Degraders (PROTACs) specifically targeting IGF2BP3 for proteasomal degradation

  • Antisense oligonucleotides or siRNAs for IGF2BP3 knockdown

  • CRISPR-based gene editing to disrupt IGF2BP3 expression in tumors

• Targeting protein-protein interactions:

  • Small molecules disrupting IGF2BP3-USP10 interaction, potentially restoring p53 stability

  • Peptide mimetics that compete with key interaction domains

  • Allosteric modulators that induce conformational changes preventing protein partner binding

• Exploiting synthetic lethality:

  • Screen for genes whose inhibition is selectively lethal in IGF2BP3-high tumors

  • Target pathways that become essential in the context of IGF2BP3 overexpression

  • Combination therapies that exploit vulnerabilities created by IGF2BP3 dependence

• Immunotherapeutic approaches:

  • Development of IGF2BP3-targeting antibody-drug conjugates

  • CAR-T cells directed against cell surface proteins upregulated by IGF2BP3

  • Cancer vaccines targeting IGF2BP3 peptides presented by tumor cells

• Targeting downstream effectors:

  • p53 pathway activators to counteract IGF2BP3's attenuation of p53 stability

  • USP10 activators to enhance p53 deubiquitination despite IGF2BP3 presence

  • Inhibitors of growth pathways activated downstream of IGF2BP3

• Biomarker-guided therapy:

  • Use IGF2BP3 expression as a stratification marker for clinical trials

  • Development of companion diagnostics to identify patients most likely to benefit

  • Monitoring IGF2BP3 levels during treatment to assess therapeutic response