Phospho-BCL2L1 (T115) Antibody

What is BCL2L1 and what role does it play in cellular processes?

BCL2L1 (also known as Bcl-x or Bcl-XL) is a member of the BCL-2 protein family that regulates apoptosis. It functions primarily as an anti-apoptotic protein by preventing mitochondrial membrane permeabilization and subsequent release of apoptotic factors. BCL2L1 interacts with pro-apoptotic proteins like BAX and BAK as well as several BH3-only proteins including BAD, BIM, and PUMA through protein-protein interactions . In normal cellular function, BCL2L1 helps maintain homeostasis by regulating the balance between cell survival and programmed cell death. The protein contains multiple conserved BCL-2 homology domains (BH1-BH4) that are essential for its anti-apoptotic function . Dysregulation of BCL2L1 expression or function has been implicated in various pathological conditions, particularly cancer development and therapeutic resistance.

What is the significance of threonine 115 (T115) phosphorylation in BCL2L1?

Threonine 115 phosphorylation represents a critical post-translational modification that regulates BCL2L1 function. This phosphorylation site (T115) is located within the protein's functional domain region (amino acids 81-130) . Phosphorylation at T115 can modulate BCL2L1's interaction with other proteins in the apoptotic pathway, potentially altering its anti-apoptotic properties. Research indicates that phosphorylation status at this site may be particularly relevant in understanding cancer mechanisms, especially in hepatocellular carcinoma (HCC) where BCL-2 family phosphorylation has been implicated in chemoresistance . The specific T115 phosphorylation has been observed to be enhanced by certain compounds, such as glycochenodeoxycholate (GCDA), which has been associated with promoting resistant HCC cells .

How does T115 phosphorylation differ from other phosphorylation sites on BCL2L1?

T115 phosphorylation occurs within a specific region of the BCL2L1 protein (amino acids 81-130) that is critical for its functional interactions . Unlike other phosphorylation sites that may primarily affect protein stability or localization, T115 phosphorylation appears to directly influence BCL2L1's binding capacity with pro-apoptotic partners. The T115 site is unique in that it is located in a region that facilitates both structural integrity and protein-protein interactions within the BCL-2 family network. Other phosphorylation sites on BCL2L1 may have distinct regulatory effects, but the T115 site has been specifically implicated in cancer contexts, particularly HCC where phosphorylation modifications contribute to treatment resistance mechanisms . This specific phosphorylation event represents a potential regulatory switch that may be targeted therapeutically in certain cancer types.

What are the validated applications for the Phospho-BCL2L1 (T115) antibody?

The Phospho-BCL2L1 (T115) antibody has been validated for several specific research applications. According to manufacturer validation data, this antibody has been confirmed effective for Enzyme-Linked Immunosorbent Assay (ELISA) and Immunohistochemistry (IHC) applications . The antibody has demonstrated specific binding to phosphorylated BCL2L1 at threonine 115 in multiple species including human, mouse, and rat samples . Validation has been performed using both phosphorylated and non-phosphorylated peptides in ELISA applications, confirming specificity for the phosphorylated form. Additionally, the antibody has been validated in IHC applications using paraffin-embedded human breast carcinoma tissues, with appropriate blocking controls using phospho-peptides demonstrating specificity . These validated applications provide researchers with reliable methods for detecting and studying this specific post-translational modification in various experimental contexts.

What are the optimal experimental conditions for using Phospho-BCL2L1 (T115) antibody in immunohistochemistry?

For optimal IHC results with the Phospho-BCL2L1 (T115) antibody, researchers should follow these methodological guidelines:

• Dilution Range: Use the antibody at a dilution of 1:100-1:300 for IHC applications .

• Tissue Preparation: Paraffin-embedded tissues have been validated with this antibody, with proper antigen retrieval techniques .

• Blocking Protocol: Include appropriate blocking steps to minimize background signal.

• Controls: Always include both positive controls (tissues known to express phosphorylated BCL2L1) and negative controls, including:

  • Antibody validation using blocking peptides (phospho-peptide competitive inhibition)

  • No primary antibody controls

  • Isotype controls

Validation images from human breast carcinoma tissue demonstrate the effectiveness of the antibody in IHC applications, particularly when paired with phospho-peptide blocking controls that confirm specificity . The protocol should be optimized for specific tissue types and fixation conditions, as these variables can affect epitope accessibility.

How can I optimize Phospho-BCL2L1 (T115) antibody usage in ELISA applications?

For ELISA applications, the following optimization procedures are recommended:

• Dilution Factor: Use a recommended starting dilution of 1:5000 for ELISA applications .

• Assay Validation: Prior to experimental samples, validate using both phospho-peptide (positive control) and non-phospho-peptide (negative control) standards .

• Signal Optimization: Titrate antibody concentrations to determine optimal signal-to-noise ratio for your specific sample types.

• Sample Preparation: Ensure consistent protein extraction methods that preserve phosphorylation status.

• Blocking: Use appropriate blocking buffers containing BSA to minimize non-specific binding.

The manufacturer's validation data demonstrates clear discrimination between phosphorylated and non-phosphorylated peptides in ELISA format , confirming the antibody's specificity. When developing phospho-specific ELISAs, it is essential to include phosphatase inhibitors throughout sample preparation to prevent loss of phosphorylation signal. Additionally, standard curves using known quantities of phosphorylated and non-phosphorylated recombinant proteins can help quantify relative phosphorylation levels in experimental samples.

How is BCL2L1 phosphorylation implicated in hepatocellular carcinoma research?

BCL2L1 phosphorylation has emerged as a significant factor in hepatocellular carcinoma (HCC) pathogenesis and treatment resistance. Research indicates that BCL-2 family proteins, including BCL2L1, are highly expressed in HCC patients and contribute to the mechanisms of chemoresistance . Specifically, phosphorylation of BCL-2 family members plays a crucial role in regulating their anti-apoptotic functions. Studies have shown that certain compounds, such as glycochenodeoxycholate (GCDA), can enhance phosphorylation at the T163 site of BCL-2 family proteins, promoting resistant HCC cells . While this specific research mentioned T163 phosphorylation, the phosphorylation at T115 may have similar functional implications in treatment resistance mechanisms.

The high mortality and poor prognosis of HCC (18% 5-year survival rate) is partially attributed to its strong invasiveness and low sensitivity to chemotherapy, with the BCL-2 family proteins playing central roles in these resistance mechanisms . The phosphorylation status of BCL2L1 may serve as both a biomarker for treatment response prediction and a potential therapeutic target in HCC management strategies.

What is the connection between BCL2L1 phosphorylation and lung cancer research?

Lung cancer research has identified significant roles for BCL2L1 and other BCL-2 family proteins in disease progression and treatment response. Abnormal expression of BCL-2 family proteins in lung cancer prevents cells with irreparable genetic changes from undergoing apoptosis, allowing their continued progression through the cell cycle and accumulation of mutations that contribute to tumorigenesis . BCL-2 family members, including BCL2L1, have been investigated as prognostic indicators for lung cancer, contributing to treatment planning and disease monitoring .

Research has demonstrated interactions between growth factor signaling pathways and BCL-2 family proteins in non-small-cell lung cancer (NSCLC). For example, the EGFR pathway can modulate the role of the BAX/BCL-2 cascade in NSCLC, with inhibition of EGFR resulting in upregulation of pro-apoptotic proteins that activate apoptotic pathways . The phosphorylation status of BCL2L1 may influence these interactions and subsequent treatment responses. Researchers studying lung cancer should consider examining phosphorylation at specific sites like T115 as potential biomarkers or therapeutic targets, particularly in the context of targeted therapy resistance mechanisms.

How does phosphorylation of BCL2L1 affect its interactions with other BCL-2 family proteins in cancer contexts?

Phosphorylation of BCL2L1 critically modifies its interactions with other BCL-2 family proteins, particularly in cancer contexts. The BCL-2 protein family includes anti-apoptotic members (BCL-2, BCL-XL, BCL-W, BCL2-A1, and MCL-1), activators (BIM and PUMA), effectors (BAX and BAK), and sensitizers (NOXA) . Phosphorylation at specific sites like T115 can alter the binding affinity of BCL2L1 for its pro-apoptotic partners, potentially enhancing or reducing its anti-apoptotic capacity.

Two primary models explain how these interactions regulate apoptosis:

• Direct Model: BH3-only proteins act as either stimulants or inhibitors. Stimulants directly activate BAX/BAK to promote apoptosis, while inhibitor BH3 proteins activate BAX/BAK by releasing BIM, tBID, and PUMA through binding to anti-apoptotic members.

• Indirect Model: BH3-only proteins bind to anti-apoptotic BCL-2 family members, releasing BAX/BAK to initiate apoptosis .

Evidence suggests these mechanisms coexist during apoptosis regulation. Phosphorylation of BCL2L1 can influence these interactions by altering binding site accessibility or affinity, thereby modulating the balance between pro-survival and pro-apoptotic signaling. In cancer contexts, aberrant phosphorylation may contribute to treatment resistance by enhancing anti-apoptotic function or disrupting normal regulatory mechanisms.

How can Phospho-BCL2L1 (T115) antibody be used to evaluate treatment efficacy in cancer models?

The Phospho-BCL2L1 (T115) antibody provides a valuable tool for evaluating treatment efficacy in cancer models through several methodological approaches:

• Treatment Response Monitoring: Researchers can use this antibody to track changes in BCL2L1 phosphorylation status before and after therapeutic interventions. This is particularly relevant for treatments targeting apoptotic pathways or upstream kinases that may influence BCL2L1 phosphorylation.

• Combination Therapy Assessment: When evaluating BCL-2 family inhibitors like venetoclax (VEN), which selectively binds to BCL-2 proteins to inhibit their anti-apoptotic function , changes in phosphorylation at T115 may provide insights into resistance mechanisms or synergistic effects.

• Methodological Approach:

  • Use IHC with the antibody (1:100-1:300 dilution) on tumor tissues collected at different treatment timepoints

  • Employ phospho-specific ELISA (1:5000 dilution) to quantify changes in phosphorylation levels in extracted proteins

  • Combine with downstream apoptotic markers (caspase activation, cytochrome C release) to correlate phosphorylation status with functional outcomes

• Biomarker Development: The phosphorylation status at T115 could potentially serve as a predictive biomarker for treatment response, particularly in cancers where BCL-2 family proteins play critical roles, such as HCC and lung cancer .

Tracking changes in this specific phosphorylation event may provide mechanistic insights into how treatments affect the apoptotic machinery within cancer cells and help identify resistance mechanisms involving post-translational modifications of BCL2L1.

What are the technical challenges in detecting BCL2L1 phosphorylation in tissue samples and how can they be overcome?

Detecting BCL2L1 phosphorylation in tissue samples presents several technical challenges that researchers must address for reliable results:

• Phosphorylation Lability: Phosphorylation modifications are highly labile and can be lost during tissue processing.

  • Solution: Immediate fixation of tissues and inclusion of phosphatase inhibitors throughout all processing steps.

• Epitope Masking: Formalin fixation can mask phospho-epitopes through protein cross-linking.

  • Solution: Optimize antigen retrieval protocols specifically for phospho-epitopes (typically using citrate buffer pH 6.0 or EDTA buffer pH 9.0 with appropriate heating conditions).

• Antibody Specificity: Ensuring phospho-specific antibody only recognizes the T115 site and not other phosphorylation sites.

  • Solution: Always include phospho-peptide blocking controls alongside experimental samples .

• Signal Quantification: Accurately quantifying phosphorylation levels in heterogeneous tissue samples.

  • Solution: Use digital image analysis with appropriate normalization to total BCL2L1 levels. Consider multiplex immunofluorescence approaches to examine multiple markers simultaneously.

• Sample Integrity: Ensuring phosphorylation status reflects in vivo conditions rather than ex vivo artifacts.

  • Solution: Minimize time between tissue collection and fixation. Consider comparing fresh frozen and formalin-fixed samples to validate findings.

When using the Phospho-BCL2L1 (T115) antibody for IHC applications, the validated protocol recommends dilutions of 1:100-1:300 , but this may require optimization for specific tissue types and fixation conditions to overcome these technical challenges.

How can I design experiments to investigate the kinase pathways responsible for BCL2L1 T115 phosphorylation?

Designing experiments to investigate kinase pathways responsible for BCL2L1 T115 phosphorylation requires a systematic approach:

• Kinase Prediction Analysis:

  • Use bioinformatic tools to predict potential kinases that might target the T115 site based on consensus sequences.

  • Examine the amino acid context around T115 (the region between amino acids 81-130) for known kinase recognition motifs.

• Kinase Inhibitor Panel Screening:

  • Treat cells with a panel of specific kinase inhibitors and measure changes in T115 phosphorylation using the Phospho-BCL2L1 (T115) antibody in ELISA (1:5000 dilution) or Western blot applications.

  • Focus on kinases implicated in cancer pathways, particularly those known to regulate apoptosis.

• Genetic Manipulation Experiments:

  • Design siRNA/shRNA knockdown or CRISPR-Cas9 knockout experiments targeting candidate kinases.

  • Overexpress constitutively active or dominant-negative kinase mutants.

  • Measure T115 phosphorylation following genetic manipulation using the antibody.

• In Vitro Kinase Assays:

  • Perform in vitro kinase assays with recombinant candidate kinases and BCL2L1 protein substrate.

  • Confirm phosphorylation using mass spectrometry and validate with the Phospho-BCL2L1 (T115) antibody.

• Pathway Stimulation/Inhibition:

  • Stimulate or inhibit upstream signaling pathways implicated in cancer (e.g., growth factor receptors, stress response pathways).

  • Monitor changes in T115 phosphorylation in response to pathway modulation.

• Correlation with Cancer Phenotypes:

  • Correlate identified kinase activity with cancer phenotypes like therapeutic resistance, particularly in HCC and lung cancer where BCL-2 family proteins play significant roles .

This experimental approach will help identify the kinases responsible for T115 phosphorylation and potentially reveal new therapeutic targets for cancers dependent on BCL2L1 anti-apoptotic function.

How should I interpret contradictory results between phospho-BCL2L1 detection and total BCL2L1 expression levels?

Contradictory results between phospho-BCL2L1 (T115) detection and total BCL2L1 expression require careful interpretation through a systematic analytical approach:

• Biological Explanations:

  • Phosphorylation-Specific Regulation: Increased phosphorylation can occur independently of total protein changes, representing activation of specific kinase pathways rather than expression changes.

  • Protein Turnover Effects: Phosphorylation may alter protein stability; some phosphorylation events increase protein degradation while others enhance stability.

  • Subcellular Redistribution: Phosphorylation can trigger relocalization of BCL2L1 without changing total levels, potentially hiding the protein from certain detection methods.

• Technical Considerations:

  • Antibody Recognition: Ensure the total BCL2L1 antibody recognition is not hindered by phosphorylation at T115.

  • Sensitivity Differences: The phospho-specific antibody may have different sensitivity compared to total protein antibodies.

  • Sample Processing Impact: Phosphorylation status is more labile than total protein; improper sample handling may selectively affect phospho-detection.

• Validation Approach:

  • Multiple Detection Methods: Confirm results using alternative techniques (e.g., if discrepancy appears in IHC, validate with ELISA) .

  • Phosphatase Controls: Treat sample aliquots with phosphatases to confirm specificity of phospho-detection.

  • Normalize Appropriately: Always calculate the ratio of phospho-BCL2L1 to total BCL2L1 rather than interpreting absolute values.

• Biological Significance Assessment:

  • Correlate with Functional Outcomes: Relate phosphorylation status to apoptotic resistance or treatment response.

  • Examine Other BCL-2 Family Members: Investigate compensatory changes in related proteins (BCL-2, BCL-W, MCL-1) .

These contradictory results may actually reflect important biological regulation and potentially reveal novel mechanisms of BCL2L1 function in cancer contexts rather than technical artifacts.

What are common false positive/negative scenarios when using Phospho-BCL2L1 (T115) antibody and how can they be mitigated?

When using the Phospho-BCL2L1 (T115) antibody, researchers should be aware of several common false positive/negative scenarios and implement appropriate mitigation strategies:

False Positive Scenarios and Mitigations:

• Cross-reactivity with Similar Phospho-epitopes:

  • Cause: The antibody may recognize similar phosphorylated motifs in other proteins.

  • Mitigation: Always include a phospho-peptide blocking control experiment . The blocking peptide competitive inhibition should eliminate specific signal while non-specific binding may remain.

• Non-specific Binding in Certain Tissues:

  • Cause: Some tissues have high endogenous biotin or phosphatases that can interfere with detection.

  • Mitigation: Use appropriate blocking reagents and include isotype control antibodies at the same concentration.

• Inadequate Blocking:

  • Cause: Insufficient blocking leads to non-specific antibody binding.

  • Mitigation: Optimize blocking conditions using BSA-containing buffers as recommended in the antibody specifications .

False Negative Scenarios and Mitigations:

• Phosphorylation Loss During Processing:

  • Cause: Phosphate groups are easily lost during sample preparation due to endogenous phosphatases.

  • Mitigation: Include phosphatase inhibitors throughout all steps of sample preparation and maintain cold temperatures.

• Epitope Masking:

  • Cause: Fixation can cross-link proteins and mask the phospho-epitope.

  • Mitigation: Optimize antigen retrieval methods specifically for phospho-epitopes.

• Antibody Concentration Issues:

  • Cause: Using improper antibody dilutions (outside the 1:100-1:300 range for IHC or 1:5000 for ELISA) .

  • Mitigation: Perform antibody titration experiments to determine optimal concentration for each specific application and tissue type.

• Insufficient Incubation Time:

  • Cause: Inadequate time for antibody binding, particularly important for phospho-epitopes.

  • Mitigation: Optimize incubation conditions, potentially using longer incubation times at 4°C.

For all applications, include positive controls (samples known to contain phosphorylated BCL2L1) and verify results using complementary approaches when possible.

How does BCL2L1 phosphorylation status influence interpretation of response to BCL-2 family inhibitors?

BCL2L1 phosphorylation status provides critical context for interpreting responses to BCL-2 family inhibitors, particularly in research and clinical applications:

• Mechanism of Resistance:

  • Phosphorylation at T115 may alter the binding affinity of BCL2L1 for BH3 mimetic drugs or change its interaction with pro-apoptotic binding partners.

  • Research has shown that phosphorylation of BCL-2 family proteins can contribute to chemoresistance mechanisms in multiple cancer types, including HCC .

  • When patients develop resistance to BCL-2 inhibitors like venetoclax (VEN) , assessment of phosphorylation status may reveal whether post-translational modifications are driving the resistance mechanism.

• Predictive Biomarker Potential:

  • Pre-treatment phosphorylation status at T115 may predict sensitivity or resistance to BCL-2 family inhibitors.

  • Changes in phosphorylation following treatment may indicate adaptive responses that precede clinical resistance.

  • Analysis should include:

    • Baseline phosphorylation assessment using the Phospho-BCL2L1 (T115) antibody in appropriate applications (IHC, ELISA)

    • Monitoring phosphorylation changes during treatment course

    • Correlation with clinical or experimental outcomes

• Combination Therapy Rationale:

  • If phosphorylation at T115 confers resistance to BCL-2 inhibitors, targeting the responsible kinase pathway could restore sensitivity.

  • Researchers should consider:

    • Identifying kinases responsible for T115 phosphorylation

    • Testing combination approaches with kinase inhibitors and BCL-2 family inhibitors

    • Measuring changes in T115 phosphorylation as a pharmacodynamic marker

• Comparative Analysis Framework:

  • Compare phosphorylation at T115 with other known regulatory phosphorylation sites on BCL2L1 and related family members

  • Consider the broader context of BCL-2 family protein interactions, including the direct and indirect models of apoptosis regulation

  • Integrate phosphorylation data with other molecular features (mutations, expression levels of interacting proteins)

This multifaceted interpretation approach provides deeper insights into treatment response mechanisms and may guide more effective therapeutic strategies targeting the BCL-2 family in cancer.

How does the function of phosphorylated BCL2L1 compare with other phosphorylated BCL-2 family proteins?

Phosphorylation of BCL-2 family proteins represents a critical regulatory mechanism with distinct functional implications across different family members:

The phosphorylation of BCL2L1 at T115 occurs within a critical functional domain (amino acids 81-130) that governs interactions with other BCL-2 family proteins. While phosphorylation of anti-apoptotic members generally enhances their protective functions, the specific mechanisms and interaction partners affected may differ. For instance, in HCC, enhanced phosphorylation of BCL-2 family proteins at various sites (including T163) has been associated with chemoresistance mechanisms , suggesting a broadly conserved role for phosphorylation in regulating apoptotic thresholds across family members, though the specific kinases and molecular consequences may vary.

What are emerging research directions regarding BCL2L1 phosphorylation in therapy resistance?

Several promising research directions are emerging regarding BCL2L1 phosphorylation in therapy resistance:

• Kinase Inhibitor Combinations:

  • Research is increasingly focusing on identifying the specific kinases responsible for T115 phosphorylation in BCL2L1.

  • Studies in HCC have already linked certain compounds like glycochenodeoxycholate (GCDA) to enhanced phosphorylation of BCL-2 family proteins, promoting resistant cancer cells .

  • Future directions include developing rational combination therapies pairing BCL-2 inhibitors like venetoclax with specific kinase inhibitors targeting the enzymes responsible for protective phosphorylation events.

• Biomarker Development:

  • Phosphorylation status at T115 and other sites may serve as predictive biomarkers for response to apoptosis-targeting therapies.

  • The Phospho-BCL2L1 (T115) antibody provides a valuable tool for developing such biomarkers in IHC and ELISA applications .

  • Research is needed to establish clinical validity through correlation with treatment outcomes across multiple cancer types.

• Novel BH3 Mimetics Development:

  • Understanding how phosphorylation affects binding to BH3-only proteins is driving the design of next-generation BH3 mimetics.

  • While venetoclax targets BCL-2 specifically, research is focusing on developing compounds that can overcome the effects of phosphorylation on binding affinity .

  • Structure-based drug design incorporating knowledge of phosphorylation effects represents a promising avenue.

• Targeted Degradation Approaches:

  • Emerging technologies like PROTACs (Proteolysis Targeting Chimeras) may overcome resistance mechanisms related to phosphorylation by targeting the protein for degradation regardless of phosphorylation status.

  • This approach could potentially address multiple resistance mechanisms simultaneously.

• Systems Biology Analyses:

  • Comprehensive analysis of the BCL-2 interactome under various phosphorylation conditions is helping to map resistance networks.

  • This approach integrates phosphoproteomic data with functional outcomes to identify critical nodes for therapeutic targeting.

These research directions highlight the importance of studying phosphorylation events like T115 in BCL2L1 for developing more effective cancer therapies and overcoming resistance mechanisms.

What methodological advances are needed to better study BCL2L1 phosphorylation in patient samples?

Several critical methodological advances are needed to enhance the study of BCL2L1 phosphorylation in patient samples:

• Improved Tissue Preservation Techniques:

  • Current tissue preservation methods often fail to adequately maintain phosphorylation status.

  • Development of specialized fixatives that better preserve phospho-epitopes while maintaining tissue architecture is needed.

  • Standardized protocols for rapid processing of patient samples with immediate phosphatase inhibitor addition would improve consistency.

• Enhanced Multiplex Detection Methods:

  • Current single-marker IHC approaches (dilutions 1:100-1:300 for the Phospho-BCL2L1 antibody) provide limited context.

  • Development of reliable multiplex immunofluorescence or mass cytometry approaches would allow simultaneous detection of:

    • Multiple phosphorylation sites on BCL2L1

    • Total BCL2L1 protein

    • Interacting partners

    • Downstream apoptotic markers

• Quantitative Digital Pathology Tools:

  • Automated image analysis algorithms specifically designed for phospho-protein quantification in heterogeneous tumor samples.

  • Standardized reporting metrics for phosphorylation levels relative to total protein expression.

  • Integration of spatial information to account for tumor heterogeneity.

• Single-Cell Phosphoproteomic Approaches:

  • Current bulk analysis methods miss the heterogeneity in phosphorylation status between individual cells.

  • Development of sensitive single-cell techniques to detect T115 phosphorylation would provide insights into resistance mechanisms at the cellular level.

  • Integration with other single-cell technologies (transcriptomics, genomics) for comprehensive profiling.

• Clinically Validated Phospho-Specific Assays:

  • Translation of research-grade antibodies like the Phospho-BCL2L1 (T115) antibody into clinically validated diagnostic assays.

  • Development of companion diagnostic tests for BCL-2 family inhibitors based on phosphorylation status.

  • Standardization of scoring systems and clinical interpretation guidelines.

These methodological advances would significantly enhance our understanding of how BCL2L1 phosphorylation contributes to cancer pathogenesis and treatment response, potentially leading to more personalized therapeutic approaches targeting this critical apoptotic regulator.