CTNNB1 Antibody

What is CTNNB1 and what makes it an important research target?

CTNNB1 (Catenin beta-1 or β-catenin) is an 85.5 kDa protein that serves dual critical functions in cells:

• As a key downstream effector in the canonical Wnt signaling pathway, where it acts as a transcriptional co-activator for TCF/LEF family transcription factors

• As a structural component of adherens junctions, where it interacts with E-cadherin to regulate cell adhesion and maintain epithelial tissue architecture

β-catenin's importance in research stems from its involvement in:

• Embryonic development and tissue homeostasis

• Neural differentiation and synaptic plasticity

• Cancer development (mutations in CTNNB1 are found in colorectal cancer, pilomatrixoma, medulloblastoma, and ovarian cancer)

• Cell growth regulation through contact inhibition signaling

The protein's dynamic regulation between membrane-associated, cytoplasmic, and nuclear pools makes it a particularly interesting target for studying cellular signaling and morphological changes .

How do I select the appropriate CTNNB1 antibody for my specific application?

Selection should be based on multiple factors aligned with your experimental needs:

Always examine validation data provided by manufacturers, including Western blots, IHC images, and knockout validation when available .

What methodological considerations are important when using CTNNB1 antibodies for immunohistochemistry?

Successful IHC with CTNNB1 antibodies requires careful attention to several methodological aspects:

• Antigen retrieval: For formalin-fixed tissues, heat-induced epitope retrieval is critical

  • Boil tissue sections in 10mM Citrate Buffer (pH 6.0) for 10-20 minutes

  • Allow to cool at room temperature for 20 minutes before proceeding

• Antibody dilution: Typically between 1:100-1:1000 for IHC-P applications, but optimization is essential for each specific antibody and tissue type

• Controls:

  • Positive control: Mouse brain, PC-12, or HeLa cells show reliable CTNNB1 expression

  • Normal adrenal tissue shows heterogeneous expression selective for zona glomerulosa and serves as an excellent control for localization patterns

• Interpretation of staining patterns:

  • Membrane staining: Indicates adherens junction localization (normal epithelial cells)

  • Cytoplasmic staining: May indicate increased protein stability

  • Nuclear staining: Often associated with active Wnt signaling and transcriptional activity

• Counterstaining: Use appropriate nuclear counterstains that won't interfere with β-catenin signal intensity

When examining CTNNB1 mutations in cancer samples, compare staining patterns with wild-type tissues, as mutations often result in aberrant nuclear accumulation that can be visualized by IHC .

How can I distinguish between different pools of β-catenin (membrane, cytoplasmic, nuclear) in my experiments?

Distinguishing between different subcellular pools of β-catenin requires specific methodological approaches:

Microscopy-based methods:

• Immunofluorescence with subcellular markers: Co-stain with E-cadherin (membrane), cytoskeletal markers (cytoplasm), and DAPI (nucleus)

• Confocal microscopy: Essential for precise localization and quantification of signal in different compartments

• Image analysis: Use software with segmentation capabilities to quantify signal intensities in different cellular compartments

Biochemical fractionation:

• Perform subcellular fractionation to isolate membrane, cytoplasmic, and nuclear fractions

• Western blot analysis of each fraction using anti-β-catenin antibody

• Include fractionation quality controls:

  • Lamin B antibody for nuclear fraction purity

  • Membrane proteins (e.g., Na+/K+ ATPase) for membrane fraction

  • Cytoskeletal proteins for cytoplasmic fraction

Functional assays:

• TCF/LEF reporter assays: Measure transcriptional activity using luciferase reporters containing TCF-4-binding sites to assess functional nuclear β-catenin

• Co-immunoprecipitation: Determine β-catenin interaction partners in different cellular compartments (E-cadherin for membrane pool, TCF/LEF for nuclear pool)

Cell density significantly affects β-catenin distribution, with higher membrane localization in confluent cells. Control for this by maintaining consistent cell density across experiments .

How can CTNNB1 antibodies be used to study Wnt signaling pathway activation?

CTNNB1 antibodies provide multiple approaches to study Wnt pathway activation:

Direct visualization methods:

• Immunofluorescence/IHC: Nuclear translocation of β-catenin is a hallmark of canonical Wnt signaling activation. Quantify nuclear/cytoplasmic ratio using image analysis software

• Cell fractionation & Western blotting: Measure β-catenin accumulation in nuclear fractions following Wnt stimulation

• Time-lapse live-cell imaging: Using SGFP2-CTNNB1 knock-in cell lines, monitor real-time changes in endogenous β-catenin localization after Wnt treatment

Biochemical and functional assessments:

• CUT&RUN and ChIP assays: Detect β-catenin binding to chromatin at Wnt target genes

  • Example alignment tracks show β-catenin binding at the AXIN2 locus, a well-established Wnt target

• TCF/LEF reporter assays: Quantify functional activation of β-catenin-dependent transcription

• Target gene expression: Measure transcription of Wnt/β-catenin target genes (e.g., AXIN2) by qRT-PCR

Experimental design considerations:

• Include positive controls such as GSK3β inhibitor CHIR99021 or purified WNT3A protein

• Monitor kinetics of response (nuclear accumulation is first statistically significant at ~30 minutes post-treatment, whereas cytoplasmic increases occur at ~45 minutes)

• Measure both total β-catenin levels and active (non-phosphorylated) β-catenin using specific antibodies

• Account for cell type-specific differences in basal Wnt pathway activity

Sophisticated biophysical approaches like Fluorescence Correlation Spectroscopy (FCS) and Number and Brightness (N&B) analysis can provide detailed information on CTNNB1-containing complexes in different cellular compartments .

What approaches can be used to validate CTNNB1 antibody specificity and performance?

Comprehensive validation of CTNNB1 antibodies requires multiple complementary approaches:

Genetic validation:

• Knockout/knockdown controls: Test antibody on CTNNB1 knockout or knockdown cells to confirm absence of signal

• CRISPR-edited cells: Use cells with fluorescently tagged endogenous CTNNB1 (e.g., SGFP2-CTNNB1) to validate antibody recognition patterns

• Allele-specific detection: In heterozygous mutant cell lines, validate antibodies can distinguish between wild-type and mutant forms

Biochemical validation:

• Western blot: Confirm single band of appropriate molecular weight (85-90 kDa)

• Immunoprecipitation followed by mass spectrometry: Verify pulled-down protein is indeed CTNNB1

• Peptide competition: Pre-incubation with immunizing peptide should abolish specific binding

• Multiple antibody comparison: Use antibodies targeting different epitopes and compare staining patterns

Functional validation:

• Subcellular localization: Verify expected distribution patterns (membrane, cytoplasmic, nuclear) under different conditions

• Response to stimuli: Confirm expected changes following Wnt pathway activation or inhibition

• Orthogonal methods: Compare results with other detection methods (e.g., fluorescent protein tagging)

Application-specific validation examples from the literature:

• For CUT&RUN applications: Alignment tracks from CUT&RUN using anti-β-catenin antibody should match ChIP-seq peaks at known targets like AXIN2

• For IHC: Compare staining patterns with known β-catenin mutations (e.g., S45F) versus wild-type tissues

Comprehensive validation should include multiple parameters as shown in commercial antibody validation data, such as comparison with known patterns, KO/KD controls, and orthogonal methods .

How do I troubleshoot non-specific or weak signals when using CTNNB1 antibodies?

When encountering issues with CTNNB1 antibody performance, systematically address these common problems:

For weak signals:

• Antibody concentration:

  • Western blot: Try increasing concentration from the recommended range (1:500-1:20000)

  • IHC/IF: Test higher concentrations (1:50-1:100) instead of dilute solutions (1:1000)

• Antigen retrieval optimization:

  • Extend boiling time in citrate buffer (up to 20 minutes)

  • Test alternative buffers (EDTA-based, pH 8-9)

  • Consider alternative retrieval methods (pressure cooker, enzymatic)

• Detection system enhancement:

  • Switch to more sensitive detection systems (e.g., TSA amplification for IHC/IF)

  • For Western blots, try longer exposure times or more sensitive chemiluminescent substrates

• Sample preparation issues:

  • Ensure proper sample fixation (over-fixation can mask epitopes)

  • Check protein extraction methods are compatible with β-catenin detection

  • For nuclear β-catenin, confirm nuclear extraction protocol is efficient

For non-specific signals:

• Blocking optimization:

  • Increase blocking time or concentration

  • Test alternative blocking agents (BSA, normal serum, commercial blockers)

  • Use additional blocking steps for endogenous peroxidase or biotin

• Antibody specificity:

  • Switch to more specific monoclonal antibodies if using polyclonals

  • Try antibodies targeting different epitopes

  • Include absorption controls with immunizing peptide

• Cross-reactivity reduction:

  • Ensure secondary antibody is appropriately specific

  • Increase washing steps (duration and number)

  • Check for potential tissue-specific autofluorescence (in IF applications)

• Sample-specific issues:

  • Cell density affects β-catenin localization; maintain consistent confluence levels

  • Wnt stimulation timing can affect results; use appropriate time points (30-45 minutes for initial response)

If troubleshooting individual parameters doesn't resolve issues, consider systematic validation using positive controls like HEK293T cells for Wnt pathway studies or known CTNNB1 mutant cell lines .

How can I use CTNNB1 antibodies to study mutations associated with cancer?

CTNNB1 mutations, particularly in the GSK3β binding domain (exon 3), are important cancer drivers. Here's how to study them using antibodies:

Detection of mutant CTNNB1 proteins:

• Immunohistochemistry approach:

  • Most CTNNB1 mutations result in protein stabilization with distinctive nuclear accumulation

  • Compare staining patterns between wild-type and mutant tissues

  • Quantify nuclear/cytoplasmic ratio as a surrogate for activating mutations

  • Example: In aldosterone-producing adenomas, all tumors with CTNNB1 mutations showed positive nuclear β-catenin staining by IHC

• Mutation-specific antibodies:

  • Engineered antibody fragments (scFvs) can be developed to specifically target common mutations

  • For example, antibodies specific to S45F mutant β-catenin peptide presented on HLA-A*03:01 have been developed

  • These can distinguish between wild-type and mutant forms based on the altered epitope

Functional analysis of mutations:

• Reporter assays:

  • Compare TCF/LEF reporter activity between wild-type and mutant CTNNB1 expression

  • Mutant proteins typically show higher transcriptional activity

• Target gene expression analysis:

  • Measure expression of Wnt target genes like AXIN2 using qRT-PCR

  • Compare between wild-type and mutant samples

• Protein interaction studies:

  • Use co-immunoprecipitation to assess altered interactions

  • Mutations often disrupt binding to destruction complex components (APC, AXIN, GSK3β)

Experimental system design:

• Isogenic cell models:

  • Use CRISPR/Cas9 to create cells with specific CTNNB1 mutations

  • Compare antibody staining between parental and mutant lines

• Clinical sample analysis:

  • Study the prevalence of CTNNB1 mutations in patient cohorts

  • Example: In a cohort of 198 aldosterone-producing adenomas, CTNNB1 mutations were detected in 5.1% of cases

• Mutation impact on therapy response:

  • Analyze how CTNNB1 mutations affect response to Wnt pathway inhibitors

  • Use antibodies to monitor β-catenin levels and localization following treatment

The study of S45F mutant β-catenin exemplifies how advanced techniques like phage display can be used to develop highly specific antibodies for detecting common oncogenic mutations .

What are important considerations when using CTNNB1 antibodies for studying protein-DNA interactions?

When using CTNNB1 antibodies for chromatin immunoprecipitation (ChIP) or CUT&RUN experiments, consider these technical aspects:

Antibody selection criteria:

• Validated for chromatin applications:

  • Not all CTNNB1 antibodies work effectively for ChIP or CUT&RUN

  • Example: ABIN2855042 is specifically validated for chromatin applications

  • Check for published CUT&RUN or ChIP-seq validation data showing enrichment at known Wnt target loci

• Epitope accessibility:

  • Choose antibodies targeting epitopes that remain accessible when β-catenin is bound to chromatin

  • N-terminal antibodies often work well as this region may be more exposed in transcriptional complexes

Experimental design considerations:

• Appropriate controls:

  • Use IgG controls from the same species as your antibody

  • Include positive controls like known β-catenin binding regions (AXIN2, c-MYC promoters)

  • Consider Wnt pathway activation (CHIR99021, WNT3A treatment) as a positive control condition

• Crosslinking optimization:

  • β-catenin interacts with DNA indirectly via TCF/LEF factors

  • Optimize formaldehyde crosslinking time (typically 10-15 minutes)

  • Consider dual crosslinking with both formaldehyde and protein-specific crosslinkers

• Sonication/digestion parameters:

  • For ChIP: Optimize sonication to generate 200-500bp fragments

  • For CUT&RUN: Carefully titrate MNase concentration and digestion time

Data analysis and validation:

• Peak identification:

  • Use appropriate peak calling algorithms (e.g., SEACR for CUT&RUN data)

  • Compare with published ChIP-seq datasets when available

• Validation approaches:

  • Confirm enrichment at known Wnt target genes

  • Example: In HEK293T cells, CUT&RUN with anti-β-catenin antibody shows clear peaks at the AXIN2 locus

  • Validate findings with orthogonal approaches (e.g., reporter assays or gene expression analysis)

• Interpretation of binding patterns:

  • β-catenin binding is often detected at enhancers and promoters of Wnt target genes

  • Co-occupancy with TCF/LEF factors is expected

  • Consider cell-type specific binding patterns

For optimal results, compare data obtained using CTNNB1 antibodies with parallel genomic approaches like ATAC-seq to correlate binding with chromatin accessibility changes upon Wnt pathway activation.

How do cell density and culture conditions affect β-catenin detection and interpretation of results?

Cell density and culture conditions significantly impact β-catenin localization and detection, creating potential confounding variables in research:

Cell density effects:

• Subcellular distribution changes:

  • At high density (confluent cells): Increased membrane localization at adherens junctions

  • At low density (sparse cells): More cytoplasmic and nuclear distribution

  • Nuclear/cytoplasmic ratio in untreated cells is approximately 0.652, showing preferential cytoplasmic localization

• Research findings demonstrating density effects:

  • When cells with mixed CTNNB1 genotypes were plated at low density, the relative abundance of mutant CTNNB1 cells increased by 235-430%

  • This growth advantage was attenuated when cells were plated at higher densities

  • In immunofluorescence studies, differences in β-catenin localization between wild-type and mutant cells were more pronounced in sparsely plated cells

Methodological controls:

• Standardization procedures:

  • Maintain consistent cell seeding density across experiments

  • Record and report confluence levels at time of analysis

  • Consider time-course experiments to track density-dependent changes

• Quantification approaches:

  • Use automated segmentation to objectively quantify subcellular distribution

  • Calculate nuclear/cytoplasmic ratios rather than absolute intensities

  • Include cell density as a variable in statistical analyses

Culture condition considerations:

• Growth factors in media:

  • Serum contains Wnt ligands that can activate β-catenin signaling

  • Use serum starvation to establish baseline β-catenin levels

  • Consider defined media for more controlled experiments

• Cell-cell contact signaling:

  • β-catenin is involved in contact inhibition

  • Different cell types exhibit varying levels of contact inhibition

  • Account for this when comparing different cell types

• Impact on experimental interpretations:

  • WNT3A treatment increases nuclear/cytoplasmic ratio from 0.652 to 1.08, indicating nuclear enrichment

  • This effect might be masked or enhanced depending on baseline cell density

  • Include density-matched controls when comparing treatments

When designing experiments involving β-catenin detection, carefully control and report cell density, and consider how it may interact with your experimental variables to affect data interpretation.

How can I accurately measure and distinguish active versus total β-catenin pools?

Distinguishing between active (signaling-competent) and total β-catenin requires specific methodological approaches:

Antibody-based discrimination:

• Phosphorylation-specific antibodies:

  • Active β-catenin lacks phosphorylation at certain residues (particularly Ser33/37/Thr41)

  • Use antibodies specifically recognizing non-phosphorylated β-catenin for active pool detection

  • Compare with total β-catenin antibodies to determine the active/total ratio

• Conformation-specific antibodies:

  • Active β-catenin may adopt distinct conformations

  • Some antibodies preferentially recognize these active conformations

Functional approaches:

• Transcriptional reporter assays:

  • pOT and pOF reporters containing TCF-4-binding sites measure functional activity

  • Normalize luciferase activity using β-galactosidase under CMV promoter control

  • This assesses the pool of β-catenin capable of driving transcription

• Co-immunoprecipitation studies:

  • Immunoprecipitate with TCF/LEF antibodies to isolate transcriptionally active β-catenin

  • Compare with total β-catenin immunoprecipitation

Biophysical characterization:

• Diffusion coefficient measurements:

  • Fluorescence Correlation Spectroscopy (FCS) can measure β-catenin mobility

  • Active β-catenin in WNT3A or CHIR99021 treated cells shows threefold increased mobility compared to controls

  • This reflects different complex formation states of active versus inactive β-catenin

• Complex size and concentration analysis:

  • Number and Brightness (N&B) analysis quantifies the concentration of β-catenin-containing complexes

  • This distinguishes between different functional pools based on complex size and abundance

Experimental design considerations:

• Positive controls:

  • GSK3β inhibitors (e.g., CHIR99021): Increase active β-catenin

  • WNT3A treatment: Physiological activation of the pathway

• Negative controls:

  • Tankyrase inhibitors: Stabilize axin and reduce active β-catenin

  • Dominant-negative TCF constructs: Block transcriptional activity

• Temporal dynamics:

  • Active β-catenin shows specific kinetics of nuclear accumulation

  • Nuclear accumulation becomes statistically significant after ~30 minutes of Wnt treatment

  • Cytoplasmic increases follow at ~45 minutes

Combining these approaches provides a comprehensive assessment of both the amount and activity state of β-catenin in your experimental system.

What advanced techniques can be used to study β-catenin mutation effects in cancer models?

Studying β-catenin mutations in cancer requires sophisticated approaches combining antibody-based methods with molecular and genetic techniques:

Generation of model systems:

• CRISPR/Cas9 engineered cell lines:

  • Create isogenic lines with specific CTNNB1 mutations (e.g., S45F, T41A)

  • Tag endogenous CTNNB1 with fluorescent proteins for live imaging

  • Example: SGFP2-CTNNB1 knock-in cell lines allow monitoring of endogenous β-catenin dynamics

• Patient-derived models:

  • Establish organoids or xenografts from tumors with known CTNNB1 mutations

  • Validate mutation status by sequencing

  • Use for testing antibody specificity and therapeutic responses

Mutation-specific detection:

• Engineered antibody approaches:

  • Development of scFv clones that selectively bind mutant peptides (e.g., S45F)

  • Example: Clone E10 specifically recognizes S45F mutant peptide bound to HLA-A*03:01

  • These can be adapted into therapeutic formats (CAR-T cells, bispecific T-cell engagers)

• MHC-I presentation analysis:

  • Study how mutant β-catenin peptides are presented on cancer cell surfaces

  • Crystal structures of wild-type and S45F mutant peptide bound to HLA-A*03:01 reveal the accessibility of the phenylalanine residue for antibody recognition

Functional characterization:

• Downstream pathway analysis:

  • Compare TCF/LEF reporter activity between wild-type and mutant cells

  • Quantify target gene expression (e.g., AXIN2 transcription)

  • Assess protein levels of Wnt targets by Western blot

• Competitive growth assays:

  • Mix wild-type and mutant cells at defined ratios

  • Track changes in proportion over time using PCR or flow cytometry

  • Example: Cells with mutant CTNNB1 show growth advantages in low-density cultures

• Three-dimensional culture systems:

  • Examine growth in soft agar to assess anchorage-independent growth

  • Analyze invasion capacity in 3D matrices

  • Evaluate morphological changes in organoid cultures

Clinical correlation:

• Mutation-phenotype relationships:

  • In APAs, CTNNB1 mutations occur in 5.1% of cases and affect "hotspot" amino acids p.Thr41 and p.Ser45

  • These mutations occur mutually exclusive from mutations in other genes (KCNJ5, ATP1A1, ATP2B3, CACNA1D)

  • Different mutations may lead to distinct histological and expression patterns

• Therapeutic implications:

  • Use antibodies to monitor response to Wnt pathway inhibitors

  • Develop mutation-specific therapeutic approaches based on antibody-derived molecules

These advanced techniques provide comprehensive insights into how CTNNB1 mutations drive cancer development and point toward potential therapeutic strategies.

How can I effectively isolate and analyze β-catenin interaction partners in different cellular compartments?

Understanding β-catenin's diverse functions requires analyzing its distinct interactomes in different cellular compartments:

Compartment-specific isolation strategies:

• Subcellular fractionation protocols:

  • Optimize fractionation to cleanly separate membrane, cytoplasmic, and nuclear pools

  • Verify fraction purity using compartment-specific markers:

    • Lamin B for nuclear fraction

    • Membrane proteins for membrane fraction

    • Cytoskeletal proteins for cytoplasmic fraction

  • Perform Western blots to confirm β-catenin distribution across fractions

• Proximity labeling approaches:

  • Generate BioID or TurboID fusion proteins with β-catenin

  • Target these constructs to specific compartments using localization signals

  • Identify compartment-specific interaction partners via biotin labeling followed by pulldown and mass spectrometry

Co-immunoprecipitation strategies:

• Antibody selection:

  • Choose antibodies that don't interfere with protein-protein interaction interfaces

  • Test multiple antibodies targeting different epitopes

  • Consider using tagged β-catenin constructs for standardized pulldowns

• Crosslinking considerations:

  • Use reversible crosslinkers to capture transient interactions

  • Optimize crosslinking conditions to maintain complex integrity without over-crosslinking

• Specialized approaches for different pools:

  • Membrane pool: Use detergent-resistant membrane fractions to isolate cadherin-catenin complexes

  • Nuclear pool: Use nuclear extracts and gentle extraction conditions to maintain transcriptional complexes

  • Destruction complex: Include phosphatase inhibitors to stabilize these interactions

Analysis of interaction partners:

• Mass spectrometry-based proteomics:

  • Use quantitative proteomics (SILAC, TMT) to compare interactomes under different conditions

  • Analyze post-translational modifications on β-catenin and partners

  • Perform correlation analysis with known interaction networks

• Validation approaches:

  • Reciprocal co-immunoprecipitation

  • Proximity ligation assay (PLA) to visualize interactions in situ

  • FRET/BRET analysis for direct interaction validation

Functional characterization:

• Impact of compartmentalization:

  • Analyze how β-catenin mutations affect interaction networks in each compartment

  • Example: Nuclear fractions from cells with disrupted mutant CTNNB1 showed significantly less β-catenin than parental cells

  • Examine how Wnt stimulation remodels the interactome in each compartment

• Dynamic regulation:

  • Track changes in interaction partners during Wnt pathway activation

  • Compare wild-type and mutant β-catenin interactomes

  • Analyze how cell density affects interaction networks

These comprehensive approaches provide insights into how β-catenin operates within distinct protein complexes to execute its diverse cellular functions.

What are the specific considerations for using CTNNB1 antibodies in live-cell imaging experiments?

Live-cell imaging of β-catenin requires specialized approaches to visualize dynamic behaviors while maintaining cell viability:

Antibody-based approaches:

• Intrabody development:

  • Convert validated CTNNB1 antibodies into intrabodies for expression inside living cells

  • Fuse with fluorescent proteins for visualization

  • Test for interference with normal β-catenin function

• Cell-permeable antibody derivatives:

  • Generate Fab fragments with cell-penetrating peptides

  • Directly label with fluorophores suitable for live imaging

  • Optimize concentration to minimize functional interference

Fluorescent protein tagging strategies:

• Endogenous tagging:

  • CRISPR/Cas9-mediated genome editing to generate clonal cell lines expressing fluorescently tagged CTNNB1

  • Example: SGFP2-CTNNB1 knock-in cells allow quantification of endogenous protein dynamics

  • Validate tagged protein behaves similarly to untagged:

    • Western blot analysis confirmed HAP1 SGFP2-CTNNB1 clones expressed only the fusion protein

    • Tagged protein responded to CHIR99021 treatment similarly to wild-type

    • Tagged protein induced target gene expression equivalently to untagged protein

• Imaging parameters:

  • Minimize phototoxicity using reduced laser power and exposure times

  • Consider spinning disk confocal for faster acquisition with less photodamage

  • Use environmental chambers to maintain physiological conditions

Quantitative analysis approaches:

• Automated cell segmentation:

  • Use computational tools to quantify dynamic subcellular distribution

  • Measure nuclear/cytoplasmic ratio changes over time

  • Example: Nuclear accumulation of CTNNB1 is favored over cytoplasmic increase after WNT3A treatment

• Advanced biophysical techniques:

  • Fluorescence Correlation Spectroscopy (FCS) to measure mobility and concentration

  • Number and Brightness (N&B) analysis for detailed information on CTNNB1-containing complexes

  • These techniques provide parameters for computational modeling of WNT/CTNNB1 signaling

• Temporal dynamics analysis:

  • First statistically significant increases in fluorescence intensity in the cytoplasm detected after ~45 min of WNT3A treatment

  • Nuclear increases first became statistically significant after ~30 min

  • Calculate and track ratio between nuclear and cytoplasmic intensities over time

Live-cell imaging reveals that endogenous CTNNB1 levels increase only 1.7-fold in the cytoplasm and 3.0-fold in the nucleus after WNT3A treatment, providing crucial quantitative parameters for understanding pathway dynamics .

How do I study post-translational modifications of β-catenin using antibodies?

Post-translational modifications (PTMs) critically regulate β-catenin stability, localization, and function. Here's how to study them effectively:

Phosphorylation analysis:

• Phosphorylation-specific antibodies:

  • Target key regulatory sites:

    • Ser33/37/Thr41: Phosphorylated by GSK3β, marks β-catenin for degradation

    • Ser45: Phosphorylated by CK1α, primes for GSK3β phosphorylation

  • Use for Western blots, IHC, IF to track specific phosphorylation status

  • Important for analyzing destruction complex activity

• Technical considerations:

  • Include phosphatase inhibitors during sample preparation

  • Use Phos-tag gels to separate phosphorylated species

  • Validate with lambda phosphatase treatment as negative control

• Mutation impact assessment:

  • S45F mutation prevents priming phosphorylation and subsequent phosphorylation

  • Compare phosphorylation patterns between wild-type and mutant cells

Other PTM detection:

• Ubiquitination analysis:

  • Co-immunoprecipitate β-catenin and blot for ubiquitin

  • Use proteasome inhibitors (MG132) to stabilize ubiquitinated forms

  • In wild-type cells without Wnt, β-catenin is ubiquitinated via BTRC and degraded by the proteasome

• Acetylation studies:

  • Use anti-acetyl-lysine antibodies following β-catenin immunoprecipitation

  • Compare acetylation status under different conditions (HDAC inhibition, Wnt activation)

• O-GlcNAcylation detection:

  • Immunoprecipitate β-catenin and blot with anti-O-GlcNAc antibodies

  • Use O-GlcNAcase inhibitors to enhance detection

PTM crosstalk analysis:

• Sequential immunoprecipitation:

  • First IP with phospho-specific antibody, then analyze other PTMs

  • Determine how one modification affects others

• Site-specific mutation studies:

  • Generate cells expressing β-catenin with mutated modification sites

  • Analyze the impact on other PTMs, stability, and function

Functional consequence assessment:

• Complex formation analysis:

  • Determine how PTMs affect interaction with destruction complex components

  • In the absence of Wnt, β-catenin forms a complex with AXIN1, AXIN2, APC, CSNK1A1, and GSK3B

  • PTMs regulate these interactions

• Subcellular localization:

  • Analyze how PTMs affect the distribution across membrane, cytoplasmic, and nuclear pools

  • Correlation between phosphorylation status and nuclear accumulation

• Transcriptional activity:

  • Use reporter assays to determine how PTMs impact transcriptional function

  • Compare wild-type, phosphomimetic, and phosphoresistant mutants