CTNND1 Antibody

What is CTNND1 and what are its key functions in cellular biology?

CTNND1 encodes the p120-catenin protein, which has multiple crucial biological functions. It maintains cell-cell junctions, regulates epithelial-mesenchymal transition, and participates in transcriptional signaling pathways . The protein is widely expressed across tissues, with notable presence in vascular endothelium . Different isoforms show tissue-specific expression patterns; for example, melanocytes and melanoma cells primarily express the long isoform 1A, whereas keratinocytes express shorter isoforms, especially 3A . CTNND1 has been implicated in various pathological conditions, including craniofacial disorders such as cleft palate, blepharocheilodontic syndrome, and several cancer types .

Which experimental methods are most appropriate for detecting CTNND1 expression?

CTNND1 can be detected through multiple complementary techniques:

• Protein detection: Western blot analysis using specific antibodies (e.g., rabbit polyclonal antibodies) is effective for quantifying CTNND1 protein levels . Immunohistochemistry (IHC) can visualize CTNND1 expression patterns in tissue sections .

• mRNA detection: Quantitative real-time PCR (qRT-PCR) measures CTNND1 transcript levels . In situ hybridization with digoxigenin-labeled antisense mRNA probes can visualize tissue-specific expression patterns .

• Functional analysis: Cell migration and invasion can be measured by Transwell and Matrigel analyses to assess the functional consequences of CTNND1 manipulation .

The optimal method depends on your specific research question. For comparing expression across different cell lines or tissues, a combination of protein and mRNA detection methods is recommended for robust validation.

What are the key considerations when selecting a CTNND1 antibody for research?

When selecting a CTNND1 antibody, researchers should consider:

• Isoform specificity: CTNND1 has multiple isoforms with tissue-specific expression. Ensure your selected antibody recognizes the relevant isoform(s) for your study. Some antibodies detect all isoforms, while others may be isoform-specific .

• Application compatibility: Verify the antibody is validated for your intended application (Western blot, IHC, etc.). For example, the AF7636 antibody is specifically validated for Western blot applications .

• Species reactivity: Confirm the antibody recognizes CTNND1 in your study species. Some antibodies, like AF7636, react with human, mouse, and rat CTNND1, with predicted reactivity to other species including pig, zebrafish, bovine, horse, and rabbit .

• Epitope information: Understanding which domain of CTNND1 the antibody recognizes is crucial, especially when studying truncated forms or specific domains .

• Validation data: Request validation data showing specificity through knockdown/knockout controls to ensure antibody specificity.

How should researchers interpret contradictory findings regarding CTNND1's role in different cancer types?

The apparent contradictory roles of CTNND1 in different cancers reflect context-dependent functions that require careful interpretation:

• Expression pattern analysis: In hepatocellular carcinoma (HCC), CTNND1 is significantly upregulated and promotes tumor progression . Conversely, in triple-negative breast cancer (TNBC), CTNND1 is downregulated in both primary tumors and bone metastases . These opposite expression patterns suggest cancer-specific roles.

• Mechanistic differences: In HCC, CTNND1 promotes metastasis by enhancing Wnt/β-catenin signaling . In TNBC, decreased CTNND1 accelerates bone metastasis through different mechanisms, including increased CXCR4 expression via PI3K/AKT pathway activation .

• Microenvironment interactions: CTNND1 knockdown in TNBC leads to increased neutrophil infiltration in the bone microenvironment, affecting cytotoxic T lymphocyte function . This demonstrates how CTNND1 can influence tumor-immune interactions differently across cancer types.

When interpreting seemingly contradictory data, researchers should consider:

• Tissue-specific isoform expression

• Interaction with different signaling pathways

• Effects on the tumor microenvironment

• Differences in experimental models

A comprehensive approach comparing signaling pathway activation, cellular phenotypes, and in vivo models across cancer types can help reconcile these differences.

What are the most effective experimental designs for studying CTNND1 function in cancer progression?

Based on published research, effective experimental designs for studying CTNND1 function include:

• Expression modulation approaches:

  • Silencing: Using shRNA or siRNA to knockdown CTNND1 expression

  • Overexpression: Transfection with CTNND1 expression vectors

  • Rescue experiments: Co-transfection with mutation-resistant CTNND1 constructs to verify specificity

  • CRISPR/Cas9: For complete knockout studies, as demonstrated in Xenopus models

• In vitro functional assays:

  • Proliferation assays to measure cell growth

  • Transwell and Matrigel analyses for migration and invasion

  • Chemotaxis assays to assess directed migration

• In vivo models:

  • Orthotopic tumor models: Directly implanting cells into the organ of origin

  • Metastasis models: Intracardiac injection for bone metastasis studies

  • Transgenic models: Conditional deletions in mice

  • SCID mice for in vivo metastasis assays

• Signaling pathway analysis:

  • Western blot to detect activation of downstream pathways (Wnt/β-catenin, PI3K/AKT)

  • Co-immunoprecipitation to identify protein-protein interactions

  • Reporter assays for transcriptional activity

• Clinical correlation:

  • Analysis of patient samples with detailed clinicopathological parameters

  • Survival analysis comparing high vs. low CTNND1 expression groups

  • Integration with public datasets (e.g., TCGA data)

How can researchers effectively analyze CTNND1's interaction with Wnt/β-catenin signaling in experimental systems?

To effectively analyze CTNND1's interaction with Wnt/β-catenin signaling:

• Protein interaction analysis:

  • Co-immunoprecipitation to detect physical interactions between CTNND1 and β-catenin

  • Proximity ligation assay to visualize protein interactions in situ

  • Subcellular fractionation to track β-catenin nuclear translocation in response to CTNND1 manipulation

• Transcriptional activity measurement:

  • TOP/FOP flash reporter assays to quantify β-catenin-mediated transcriptional activity

  • qRT-PCR analysis of Wnt target genes like Cyclin D1, WNT11, and MMP7

  • Chromatin immunoprecipitation to assess β-catenin binding to target gene promoters

• Pathway manipulation:

  • Use of Wnt pathway activators (e.g., GSK3β inhibitors) or inhibitors in combination with CTNND1 modulation

  • Rescue experiments with constitutively active β-catenin in CTNND1-depleted cells

• Correlation analysis in clinical samples:

  • Multiplex immunohistochemistry for co-localization of CTNND1 with β-catenin

  • Expression correlation between CTNND1 and Wnt targets in patient samples

Research has demonstrated that CTNND1 expression positively correlates with β-catenin, WNT11, Cyclin D1, and BMP7/MMP7 expression in human HCC specimens . This correlation supports the functional relationship between CTNND1 and Wnt signaling in cancer progression.

What controls should be included when using CTNND1 antibodies for Western blot analysis?

When performing Western blot analysis with CTNND1 antibodies, include these essential controls:

• Positive controls:

  • Cell lines with known high CTNND1 expression (e.g., invasive HCC cell lines for positive control)

  • Recombinant CTNND1 protein (if available)

• Negative controls:

  • CTNND1 knockdown or knockout cell lines

  • Cell lines with naturally low CTNND1 expression (e.g., non-invasive HCC cell lines)

• Specificity controls:

  • Peptide competition assay: Pre-incubating the antibody with immunizing peptide should abolish specific signals

  • Multiple antibodies targeting different epitopes of CTNND1 to confirm band identity

  • Different isoform-expressing cells to verify isoform-specific detection

• Loading controls:

  • Housekeeping proteins (β-actin, GAPDH) to normalize protein loading

  • Total protein staining (Ponceau S, SYPRO Ruby) as an alternative normalization method

• Molecular weight verification:

  • CTNND1 has a predicted molecular weight of approximately 108 kDa , but observed weight may vary due to post-translational modifications

  • When examining multiple isoforms, reference the expected weight of each isoform

How can researchers optimize immunohistochemical detection of CTNND1 in tissue samples?

For optimal immunohistochemical detection of CTNND1:

• Sample preparation:

  • Proper fixation is critical; overfixation can mask epitopes

  • For formalin-fixed paraffin-embedded (FFPE) samples, optimize antigen retrieval methods (heat-induced or enzymatic)

  • Consider using both FFPE and frozen sections for validation

• Antibody optimization:

  • Perform titration experiments to determine optimal antibody concentration

  • Test multiple antibodies targeting different CTNND1 epitopes

  • Validate antibody specificity using CTNND1 knockdown/knockout tissues

• Detection system selection:

  • For low expression, use amplification systems like tyramide signal amplification

  • For co-localization studies, consider multiplex IHC or immunofluorescence

• Controls:

  • Positive controls: Tissues known to express CTNND1 (e.g., vascular endothelium)

  • Negative controls: Primary antibody omission and isotype controls

  • Comparison with mRNA in situ hybridization for validation

• Scoring system development:

  • Establish a standardized scoring system based on staining intensity and percentage of positive cells

  • Consider digital image analysis for objective quantification

  • Potential categorization: "low CTNND1 expression" versus "high CTNND1 expression" groups for statistical analysis

How should researchers address variability in CTNND1 detection across experimental replicates?

Variability in CTNND1 detection may stem from several sources. Address these systematically:

• Biological variability:

  • Cell density effects: CTNND1 expression can vary with cell confluence due to its role in cell-cell adhesion

  • Cell passage number: Expression may change with extended culturing

  • Solution: Standardize cell culture conditions and document passage numbers

• Technical variability:

  • Antibody lot-to-lot variation

  • Inconsistent sample preparation

  • Solution: Use the same antibody lot when possible; implement standardized protocols

• Isoform complexity:

  • Multiple CTNND1 isoforms exist with tissue-specific expression patterns

  • Solution: Use isoform-specific primers for qRT-PCR; select antibodies that detect relevant isoforms

• Post-translational modifications:

  • Phosphorylation status can affect antibody binding

  • Solution: Consider phospho-specific antibodies if relevant

• Statistical approach:

  • Perform sufficient biological replicates (minimum n=3)

  • Use appropriate statistical tests to determine significance

  • Consider normalization methods carefully

What are the key considerations when interpreting CTNND1 expression data in relation to patient outcomes?

When correlating CTNND1 expression with patient outcomes:

• Stratification approach:

  • Define clear criteria for "high" versus "low" expression based on quantitative measurements

  • Consider using median split, quartiles, or optimized cutoff values

  • Evaluate the robustness of findings across different stratification methods

• Statistical analysis:

  • Use Kaplan-Meier survival analysis with log-rank tests to compare survival between groups

  • Perform multivariate analysis (Cox regression) to adjust for confounding factors

  • Report hazard ratios with confidence intervals

• Clinical correlation:

  • Analyze associations between CTNND1 expression and clinicopathological parameters

  • Research has shown significant correlations between CTNND1 expression and tumor diameter, microvascular invasion, and tumor differentiation in HCC

  • Consider cancer-specific parameters (e.g., HBsAg status in HCC, hormone receptor status in breast cancer)

• Tissue heterogeneity:

  • Account for intratumoral heterogeneity by examining multiple regions

  • Consider expression in tumor versus stroma

  • For metastasis studies, compare expression between primary tumors and metastatic lesions

• Validation approaches:

  • Validate findings in independent cohorts

  • Compare with public datasets (e.g., TCGA data)

  • Consider meta-analysis if multiple smaller studies exist

What emerging technologies could enhance CTNND1 research in cancer biology?

Several emerging technologies hold promise for advancing CTNND1 research:

• Single-cell technologies:

  • Single-cell RNA sequencing to identify cell-specific CTNND1 expression patterns within heterogeneous tumors

  • Single-cell proteomics to analyze CTNND1 protein levels and modifications at the individual cell level

  • Spatial transcriptomics to map CTNND1 expression within the tumor microenvironment

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize CTNND1 localization at subcellular structures

  • Intravital imaging to monitor CTNND1 dynamics during metastasis in live animal models

  • Multiplexed imaging (e.g., CODEX, MIBI) to simultaneously detect multiple proteins in the CTNND1 pathway

• Gene editing advancements:

  • CRISPR activation/inhibition systems for temporal control of CTNND1 expression

  • Base editing or prime editing for introducing specific CTNND1 mutations

  • CRISPR screens to identify synthetic lethal interactions with CTNND1

• Liquid biopsy applications:

  • Monitoring circulating tumor DNA for CTNND1 alterations as potential biomarkers

  • Analyzing CTNND1 expression in circulating tumor cells

  • Exosomal CTNND1 mRNA as a potential non-invasive biomarker

• In silico approaches:

  • AI/machine learning to predict CTNND1 interaction networks

  • Structural biology and molecular dynamics simulations to understand CTNND1 protein interactions

  • Systems biology approaches to model CTNND1's role in cellular signaling networks

How might CTNND1 research translate into clinical applications?

CTNND1 research shows potential for clinical translation in several areas: