bcl9l Antibody

What is BCL9L and why is it important in cancer research?

BCL9L (B-cell CLL/lymphoma 9-like protein) is a transcriptional regulator that acts as an activator in the Wnt/β-catenin signaling pathway. It promotes beta-catenin transcriptional activity and plays a significant role in tumorigenesis . BCL9L is structurally similar to BCL9, with both functioning as co-factors in the Wnt pathway.

The importance of BCL9L in cancer research stems from several key observations:

• BCL9L expression is significantly upregulated in multiple cancer types, including pancreatic ductal adenocarcinoma (PDAC), colorectal cancer, and bladder cancer

• It enhances the neoplastic transforming activity of CTNNB1 (β-catenin)

• BCL9L has been linked to tumor invasion, metastasis, and resistance to therapy

• Recent studies show that targeting BCL9/BCL9L can enhance antigen presentation and boost immune responses against cancer

Methodologically, when investigating BCL9L in cancer contexts, researchers should consider both its transcriptional activity and its role in regulating cell behaviors like proliferation, migration, and invasion.

What are the key differences between BCL9L antibodies available for research applications?

When selecting BCL9L antibodies, researchers should consider:

• Target region: Some antibodies target N-terminal vs. C-terminal regions, which may affect detection depending on protein modifications or interactions

• Validated applications: Not all antibodies work equally well across applications; select those validated for your specific experimental context

• Species reactivity: While many BCL9L antibodies are human-specific, some cross-react with mouse and rat orthologs

• Controls: Using appropriate positive controls (e.g., HeLa cell lysate for many BCL9L antibodies)

What experimental applications can BCL9L antibodies be used for?

BCL9L antibodies have been validated for multiple experimental applications:

• Western Blot (WB): For detecting BCL9L protein (approximately 157 kDa) in cell and tissue lysates

• Immunohistochemistry (IHC): For examining BCL9L expression and localization in formalin-fixed paraffin-embedded (FFPE) tissues

• Immunocytochemistry (ICC)/Immunofluorescence (IF): For visualizing subcellular localization of BCL9L

• Flow Cytometry (FCM): For analyzing BCL9L expression in cell populations

• Immunoprecipitation (IP): For studying protein-protein interactions, particularly with β-catenin

• ELISA: For quantitative detection of BCL9L in various sample types

Methodology note: When using BCL9L antibodies for subcellular localization studies, it's critical to recognize that BCL9L distribution can vary by cell type. For example, BCL9L has been observed predominantly in the nucleus of Cal29 cells but mainly in the cytoplasm of T24 cells .

How should researchers optimize BCL9L antibody conditions for Western blot analysis?

Optimizing BCL9L antibody conditions for Western blot requires systematic approach:

• Sample preparation:

  • Use appropriate lysis buffers that preserve protein integrity

  • Include protease inhibitors to prevent degradation

  • Recommended positive controls: HeLa, HCT-116, K562, or HEK-293 cell lysates

• Electrophoresis and transfer conditions:

  • Given BCL9L's high molecular weight (approximately 157 kDa), use lower percentage gels (6-8%)

  • Increase transfer time for large proteins

• Antibody dilution optimization:

  • Start with manufacturer-recommended dilutions (typically 1:500-1:1000)

  • Test a dilution series if signal-to-noise ratio is suboptimal

  • For monoclonal antibodies like 3B9C1, 1:500 dilution has been validated

  • For polyclonal antibodies, 1 μg/mL has been effective in detecting BCL9L in cancer cell lines

• Detection system selection:

  • Enhanced chemiluminescence (ECL) is commonly used

  • For weaker signals, consider more sensitive detection systems like SuperSignal West Femto

• Optimization for specific research contexts:

  • When studying BCL9L knockdown effects, confirm reduced expression using validated antibodies

  • For studies involving β-catenin interactions, consider membrane fractionation protocols to distinguish localization patterns

What are the critical methodological considerations for immunohistochemical detection of BCL9L in cancer tissues?

Detecting BCL9L in cancer tissues requires careful methodological considerations:

• Tissue preparation and antigen retrieval:

  • FFPE tissue sections (typically 4-6 μm thickness)

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) is generally effective

  • Ensure complete deparaffinization to prevent nonspecific binding

• Blocking and antibody incubation:

  • Thorough blocking (e.g., 5-10% normal serum from the species of secondary antibody)

  • Validated dilutions: 1:200 for monoclonal antibodies like 3B9C1

  • Overnight incubation at 4°C often improves specific staining

• Detection systems:

  • DAB (3,3'-diaminobenzidine) is commonly used for chromogenic detection

  • For multiplex staining, consider fluorescent-based detection systems

• Result interpretation:

  • BCL9L expression is heterogeneous in tumors and even in non-dysplastic urothelium

  • Nuclear staining is typically stronger when BCL9L expression is high

  • In papillary tumors, peripheral cell layers often express more BCL9L than inner tumor layers

• Quantification approaches:

  • H-score method has been applied to quantify BCL9L expression

  • Digital image analysis can provide more objective quantification

Research observation: In bladder cancer studies, BCL9L staining was observed in both nucleus and cytoplasm but was strongly intensified in the nucleus, especially with high expression. Non-dysplastic urothelium showed increased expression toward superficial cell layers .

How can researchers validate BCL9L antibody specificity and ensure reproducible results?

Validating BCL9L antibody specificity is crucial for reliable experimental outcomes:

• Genetic validation approaches:

  • siRNA/shRNA knockdown: Compare BCL9L staining/detection in knockdown vs. control samples

  • Rescue experiments: Re-express BCL9L in knockdown cells to restore antibody reactivity

  • CRISPR/Cas9 knockout: Eliminate BCL9L expression to confirm antibody specificity

• Biochemical validation methods:

  • Blocking peptide experiments: Pre-incubate antibody with immunizing peptide before detection

  • Multiple antibody validation: Use different antibodies targeting distinct epitopes

  • Western blot analysis should show a band at the expected molecular weight (~157 kDa)

• Controls for different applications:

  • Positive tissue controls: Colorectal, pancreatic, or cervical cancer tissues with known BCL9L expression

  • Negative controls: Omit primary antibody or use isotype control

  • Subcellular localization controls: Compare with established localization patterns

• Reproducibility considerations:

  • Document detailed protocols including antibody catalog numbers, lots, dilutions

  • Include internal reference standards across experiments

  • For polyclonal antibodies, consider purchasing larger lots to minimize batch variation

Experimental example: In a study validating BCL9L knockdown, both mRNA and protein levels were assessed. The mRNA level of BCL9L was significantly reduced in Cal29 (3-fold and 1.5-fold on day 2 and 3, respectively) and T24 (1.8-fold and 1.6-fold on day 2 and 3, respectively) after knockdown compared to siControl (p < 0.05) .

How can BCL9L antibodies be utilized to investigate the Wnt/β-catenin signaling pathway?

BCL9L antibodies can provide valuable insights into Wnt/β-catenin signaling through multiple experimental approaches:

• Co-immunoprecipitation (Co-IP) studies:

  • Use BCL9L antibodies to immunoprecipitate complexes and probe for β-catenin

  • Alternatively, immunoprecipitate with β-catenin antibodies and detect BCL9L

  • This approach has revealed increased retention of β-catenin at the plasma membrane in BCL9L knockdown cells

• Chromatin immunoprecipitation (ChIP):

  • Use BCL9L antibodies to identify genomic regions where BCL9L-containing transcriptional complexes bind

  • Can be coupled with sequencing (ChIP-seq) for genome-wide analysis

• Immunofluorescence co-localization:

  • Double staining with BCL9L and β-catenin antibodies reveals their spatial relationship

  • In BCL9L knockdown studies, increased retention of β-catenin at the plasma membrane has been observed

• Pathway modulation studies:

  • Combine BCL9L antibody detection with Wnt pathway activators like SKL2001

  • Research has shown that SKL2001 increases expression of Wnt/β-catenin target genes (AXIN2, LEF1) in a manner that can be reduced by BCL9L knockdown in certain cell lines

• Target gene expression analysis:

  • Correlate BCL9L detection with expression of Wnt target genes:

    • AXIN2, LEF1 (canonical pathway markers)

    • SP5, BIRC5, MMP9, MMP14 (cancer-related targets)

Research finding: Studies have revealed cell-type specific effects, where BCL9L knockdown reduced Wnt/β-catenin target gene expression in Cal29 cells but not in T24 cells, suggesting context-dependent roles for BCL9L in Wnt signaling .

What methodological approaches can be used to study BCL9L's role in tumor progression and metastasis?

Investigating BCL9L's role in tumor progression requires multiple methodological approaches:

• Expression correlation studies:

  • Compare BCL9L levels across cancer stages and grades using antibody-based detection

  • In bladder cancer, BCL9L staining was observed to be heterogeneous but showed increased expression in dysplastic urothelium compared to non-dysplastic areas

  • Quantitative analysis revealed significantly higher BCL9L expression in PDAC (mean score 9.6) compared to normal duct cells (mean score 3.16)

• Functional assays after BCL9L modulation:

  • Proliferation assays: Crystal violet staining showed BCL9L knockdown significantly represses proliferation of bladder cancer cell lines

  • Migration assays: Real-time cell analysis with the xCELLigence system demonstrated reduced migration in BCL9L knockdown cells

  • Invasion assays: Matrigel-coated chambers revealed BCL9L knockdown significantly represses invasion of Cal29 and T24 cells

• EMT marker analysis:

  • BCL9L knockdown has been shown to provoke an increment of E-cadherin protein levels

  • Monitor epithelial-mesenchymal transition markers in relation to BCL9L expression

• In vivo models:

  • Xenograft models have confirmed the relevance of BCL9L for tumor growth

  • Pharmacological inhibition of BCL9/BCL9L with inhibitors like hsBCL9z96 has shown delayed tumor growth

• Immune response studies:

  • Recent research shows targeting BCL9/BCL9L enhances antigen presentation by stimulating conventional type 1 dendritic cell (cDC1) activation

  • Single-cell transcriptomics analysis has revealed that Bcl9/Bcl9l deficient cDC1 were superior to wild-type cDC1 at activation and antigen presentation via NF-κB/IRF1 signaling

Experimental observation: In functional studies, BCL9L knockdown did not affect apoptosis of bladder cancer cells as measured by flow cytometry with annexin V-FITC and propidium iodide staining, suggesting its effects on tumor progression are primarily through other mechanisms .

How do you differentiate between BCL9 and BCL9L in experimental studies?

Distinguishing between BCL9 and BCL9L requires careful experimental design and interpretation:

• Antibody selection strategies:

  • Use antibodies specifically validated against the unique regions of BCL9 or BCL9L

  • Verify specificity by testing against recombinant proteins for each homolog

  • Consider using antibodies raised against regions with low sequence homology

• Genetic approaches for specific targeting:

  • Design siRNA/shRNA sequences that target unique regions of each gene

  • Validate knockdown specificity by measuring both BCL9 and BCL9L expression

  • For CRISPR/Cas9 targeting, design guide RNAs specific to each gene

• Expression analysis discrimination:

  • At the mRNA level: Use gene-specific primers for RT-PCR/qPCR

  • At the protein level: Western blot analysis should show distinct bands (BCL9: ~149.3 kDa, BCL9L: ~157 kDa)

• Functional distinctions:

  • While both proteins interact with β-catenin, they may have context-dependent effects

  • BCL9 and BCL9L are considered evolutionary duplicates of Legless that perform similar tasks but with different regulatory mechanisms

• Understanding overlapping vs. distinct functions:

  • In some contexts, targeting both BCL9 and BCL9L may be necessary

  • Recent research on antigen presentation examined both BCL9/BCL9L knockout mice and pharmacological inhibition affecting both proteins

Research insight: While both proteins serve as co-factors in the Wnt pathway, their expression patterns and precise functions may differ across tissue types and disease contexts. Studies targeting both proteins (BCL9/BCL9L) have shown enhanced antitumor responses, suggesting collaborative or redundant roles in some contexts .

What are the common technical challenges when using BCL9L antibodies and how can they be addressed?

Researchers frequently encounter several challenges when working with BCL9L antibodies:

• High molecular weight detection issues:

  • Problem: Poor transfer of high molecular weight BCL9L (~157 kDa) in Western blots

  • Solution: Use lower percentage gels (6-8%), extend transfer time, add SDS to transfer buffer

• Subcellular localization variability:

  • Problem: Inconsistent nuclear vs. cytoplasmic staining across cell types

  • Solution: Use cell fractionation controls; recognize that BCL9L localization is cell-type specific (e.g., predominantly nuclear in Cal29 cells but mainly cytoplasmic in T24 cells)

• Signal specificity concerns:

  • Problem: Multiple bands or unexpected staining patterns

  • Solution: Validate with knockdown controls; use blocking peptides; compare multiple antibodies targeting different epitopes

• Background in IHC/ICC:

  • Problem: High background staining obscuring specific signal

  • Solution: Optimize blocking conditions; titrate antibody concentration; include adequate washing steps

• Reproducibility between experiments:

  • Problem: Variation in staining intensity or pattern across experiments

  • Solution: Standardize protocols; include internal controls; for polyclonal antibodies, consider purchasing larger lots

• Cross-reactivity with BCL9:

  • Problem: Potential cross-reactivity with the BCL9 homolog

  • Solution: Validate antibody specificity using recombinant proteins or cells with known expression of each protein

Technical note: When performing co-immunoprecipitation experiments to study BCL9L-β-catenin interactions, consider using nanobody-based approaches that have been successful in previous studies .

How should researchers interpret BCL9L expression patterns in different cancer contexts?

Interpreting BCL9L expression requires consideration of several context-dependent factors:

• Heterogeneous expression patterns:

  • BCL9L expression is often heterogeneous within tumors and across tumor types

  • In papillary tumors, peripheral cell layers often express more BCL9L than inner tumor layers

  • Expect variation even within the same tumor sample

• Subcellular localization significance:

  • Nuclear localization often correlates with transcriptional activity

  • Cytoplasmic localization may indicate sequestration or alternative functions

  • In non-dysplastic urothelium, expression increases toward superficial cell layers

• Correlation with clinical parameters:

  • Elevated BCL9L expression correlates with higher nuclear grade cancer phenotype in some contexts

  • In breast cancer, BCL9L expression has been associated with ErbB2/HER-2 expression

  • PDAC tissues show significantly higher BCL9L expression compared to chronic pancreatitis and normal pancreas tissues

• Integration with pathway markers:

  • Interpret BCL9L expression in conjunction with β-catenin localization

  • Consider correlation with Wnt target gene expression (AXIN2, LEF1, etc.)

  • Relationship to E-cadherin expression may indicate EMT involvement

• Quantification approaches:

  • H-score method can provide semi-quantitative assessment of expression levels

  • Consider reporting both intensity and percentage of positive cells

  • Digital pathology approaches may provide more objective quantification

Research finding: In bladder cancer studies, BCL9L was expressed very heterogeneously in tumor samples as well as in dysplastic and non-dysplastic urothelium, with significantly higher expression observed in MIBC compared to matched NMIBC samples .

What controls are essential when conducting BCL9L functional studies using antibodies?

Robust controls are critical for reliable BCL9L functional studies:

• Genetic manipulation controls:

  • Non-targeting siRNA/shRNA controls should be carefully selected

  • Consider rescue experiments where BCL9L is re-expressed in knockdown cells

  • For double knockout studies (BCL9/BCL9L), single knockout controls are essential

• Functional assay controls:

  • For proliferation assays: Include growth curve controls under standard conditions

  • For migration/invasion: Compare to established high/low motility cell lines

  • For immunoprecipitation: Include IgG control and input samples

• Pharmacological inhibition controls:

  • When using BCL9/BCL9L inhibitors like hsBCL9z96, include dose-response curves

  • Test inhibitor effects on known BCL9L-negative systems to confirm specificity

  • Compare pharmacological inhibition with genetic knockdown approaches

Research example: Studies investigating BCL9L's role in Wnt signaling have included controls with both Wnt pathway activators (SKL2001) and BCL9L knockdown, allowing researchers to determine whether BCL9L is required for pathway activation in specific cellular contexts .

How can researchers address conflicting results when studying BCL9L across different cell lines or cancer types?

Addressing conflicting results requires systematic investigation of potential sources of variation:

• Cell-type dependent effects:

  • BCL9L function appears to be context-dependent

  • Example: BCL9L knockdown reduced Wnt/β-catenin target gene expression in Cal29 cells but not in T24 cells

  • Solution: Test hypotheses across multiple cell lines representing different cancer subtypes

• Baseline expression level considerations:

  • Characterize endogenous BCL9L levels before manipulation

  • Higher vs. lower expressing cell lines may respond differently to knockdown

  • Document both mRNA and protein expression levels across experimental models

• Pathway context variations:

  • Assess Wnt pathway activation status in different models

  • Different cancer types may have distinct β-catenin mutation profiles

  • Consider parallel pathway interactions that may compensate for BCL9L loss

• Methodological reconciliation approaches:

  • Standardize experimental conditions across cell lines

  • Use multiple methodologies to confirm observations (e.g., complement knockdown with CRISPR knockout or inhibitor studies)

  • Conduct side-by-side comparisons under identical conditions

• Integrative analysis strategies:

  • Correlate in vitro findings with patient data

  • Perform comprehensive pathway analysis including multiple components

  • Consider single-cell approaches to address heterogeneity

Research insight: Studies have shown that BCL9L can be localized differently across cell types - predominantly nuclear in Cal29 cells but mainly cytoplasmic in T24 and TCCsup cells . This differential localization may contribute to context-dependent functional outcomes and should be considered when interpreting conflicting results.

How are BCL9L antibodies being used to develop or evaluate therapeutic strategies targeting the Wnt pathway?

BCL9L antibodies are playing crucial roles in therapeutic development:

• Inhibitor development and validation:

  • Antibodies are used to confirm target engagement of BCL9/BCL9L inhibitors like hsBCL9z96

  • Western blotting and immunohistochemistry provide evidence of pathway modulation after inhibitor treatment

• Combination therapy assessment:

  • BCL9L antibodies help evaluate the effects of combining Wnt pathway inhibition with other therapeutic approaches

  • Recent research suggests potential synergy between BCL9/BCL9L targeting and immunotherapy approaches

• Biomarker development:

  • BCL9L detection may serve as a potential biomarker for patient stratification

  • Antibody-based assays can potentially identify patients most likely to benefit from Wnt pathway targeting

• Mechanism-of-action studies:

  • Antibodies reveal how targeting BCL9/BCL9L affects downstream signaling

  • Research has shown that targeting BCL9/BCL9L enhances antigen presentation by stimulating cDC1 activation and infiltration into tumors

• Resistance mechanism investigation:

  • BCL9L antibodies can help identify adaptations that occur after treatment

  • Changes in localization or expression may indicate pathway rewiring