Phospho-BCL2 (T74) Antibody

What is Phospho-BCL2 (T74) Antibody and what epitope does it recognize?

Phospho-BCL2 (T74) Antibody is a rabbit polyclonal antibody specifically designed to detect the BCL2 protein only when it is phosphorylated at the threonine 74 (T74) position. This antibody is developed using synthesized phospho-peptide derived from human BCL2 protein around the T74 phosphorylation site . It serves as a crucial tool for researchers studying post-translational modifications of BCL2 in relation to apoptotic regulation and cellular stress responses.

The antibody specifically recognizes the endogenous levels of BCL2 protein when phosphorylated at T74, making it valuable for studying this particular post-translational modification without cross-reactivity to non-phosphorylated BCL2 or other phosphorylation sites . This specificity enables researchers to investigate the unique role of T74 phosphorylation in BCL2's function.

Why is studying BCL2 phosphorylation at T74 important in apoptosis research?

Studying BCL2 phosphorylation at T74 is particularly important because BCL2 is a central regulator of the intrinsic apoptotic pathway. BCL2 functions by binding and neutralizing pro-apoptotic proteins including mitochondrial permeabilizers Bax and Bak, as well as cellular stress sensors like Bim, Bid, Puma, Bad, Bmf, and in some conditions, Noxa .

Post-translational modification by phosphorylation represents a critical regulatory mechanism for BCL2's anti-apoptotic function. While BCL2 expression alone might not be sufficient to protect cells from apoptosis in all physiological contexts, its phosphorylation status—including at T74—creates a dynamic and reversible system to rapidly regulate BCL2 activity and affect cell viability . This phosphorylation occurs within BCL2's flexible loop domain (FLD), a natively disordered region that bridges the BCL2 homology motifs BH3 and BH4 .

Unlike the extensively studied S70 phosphorylation site (which is required for full anti-apoptotic function), the specific role of T74 phosphorylation is still being elucidated, making the T74 phospho-specific antibody a valuable research tool.

What experimental applications is Phospho-BCL2 (T74) Antibody validated for?

The Phospho-BCL2 (T74) Antibody has been validated for several experimental applications that allow researchers to detect and quantify phosphorylation at this specific residue:

While these are the validated applications, researchers should note that optimal dilutions may vary depending on experimental conditions and should be determined empirically. The antibody may potentially be applicable to other techniques like western blotting and immunofluorescence, though specific validation data for these applications was not provided in the search results .

How should Phospho-BCL2 (T74) Antibody be stored and handled to maintain its activity?

For optimal performance and longevity of the Phospho-BCL2 (T74) Antibody, proper storage and handling are essential:

• Storage temperature: Store at -20°C or -80°C upon receipt .

• Avoid repeated freeze-thaw cycles which can degrade antibody quality and affect binding specificity .

• The antibody is provided in liquid form in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide, which helps stabilize the antibody during storage .

• Working dilutions should be prepared fresh before use and can typically be stored at 4°C for short periods (1-2 weeks).

• When handling, use sterile techniques to prevent contamination, as microbiological contaminants can degrade the antibody.

Following these guidelines will help ensure consistent experimental results and maximize the useful life of the antibody reagent.

How does phosphorylation at T74 compare with other phosphorylation sites on BCL2 in terms of functional significance?

BCL2 undergoes phosphorylation at multiple sites within its flexible loop domain (FLD), including T56, S70, T74, and S87, each potentially having distinct functional implications . Current research reveals significant differences between these phosphorylation events:

• S70 phosphorylation: Well-characterized and known to be required for BCL2's full anti-apoptotic function, particularly in murine IL-3-dependent myeloid cell lines . This is considered a single-site phosphorylation event that enhances BCL2's protective capacity.

• T74 phosphorylation: Often occurs as part of multi-site phosphorylation events in response to microtubule-targeting drugs like paclitaxel and colchicine . Unlike S70 phosphorylation which enhances BCL2's anti-apoptotic function, multi-site phosphorylation involving T74 may actually inhibit BCL2's protective function .

• S87 phosphorylation: Research indicates this is a primary target for kinases like JNK and ERK2, suggesting some sequence or structural specificity for phosphorylation by these kinases . Molecular dynamics simulation studies have shown that phosphorylation at S87 induces conformational changes in the peptide structure .

These differences highlight the complex regulatory network governing BCL2 function. While phosphorylation at S70 appears to enhance BCL2's anti-apoptotic activity, the role of T74 phosphorylation may be more context-dependent, potentially serving as part of a multi-site phosphorylation pattern that modulates BCL2 function differently than single-site phosphorylation events .

What kinases are responsible for phosphorylating BCL2 at T74, and how does this differ from other sites?

The phosphorylation of BCL2 at different sites involves specific kinases with varying preferences and activities:

• S87 appears to be the primary phosphorylation site for both JNK and ERK2, suggesting some sequence or structural specificity for these kinases .

• T74 phosphorylation specificity is less clearly defined in the available research. While JNK has been implicated in multi-site BCL2 phosphorylation following treatment with microtubule-targeting drugs, the direct kinase-substrate relationship for T74 specifically is not as well characterized as for S87 .

• T56 phosphorylation has been associated with different cellular stressors, but the kinases involved may differ from those phosphorylating T74.

Researchers investigating T74 phosphorylation specifically should consider:

• The potential for sequential phosphorylation, where modification at one site influences the likelihood of phosphorylation at others

• The role of phosphatases in dynamically regulating BCL2 phosphorylation status

• The context-dependence of kinase activity, as different stimuli may activate different kinases leading to distinct phosphorylation patterns

This site-specific phosphorylation contributes to the complex regulation of BCL2's anti-apoptotic function in response to various cellular conditions.

How can researchers distinguish between single-site and multi-site phosphorylation events on BCL2 in experimental settings?

Distinguishing between single-site and multi-site phosphorylation of BCL2 is methodologically challenging but critical for understanding the functional implications. Researchers can employ several complementary approaches:

The choice between these methods depends on the specific research question, with most rigorous studies employing multiple complementary approaches.

What controls should be included when using Phospho-BCL2 (T74) Antibody in immunohistochemistry or ELISA experiments?

When designing experiments with Phospho-BCL2 (T74) Antibody, including appropriate controls is essential for result validation and interpretation:

Essential Controls for Immunohistochemistry:

• Positive control: Tissue samples known to express phosphorylated BCL2 at T74, such as certain lymphoma samples or cell lines treated with microtubule-targeting drugs that induce BCL2 phosphorylation .

• Negative control: Samples known not to express phosphorylated BCL2 at T74, or samples treated with phosphatase to remove phosphorylation.

• Antibody controls:

  • Primary antibody omission to assess background staining

  • Isotype control (rabbit IgG at equivalent concentration) to evaluate non-specific binding

• Phosphorylation specificity controls:

  • Pre-treatment of some sections with phosphatase to demonstrate phosphorylation-dependent binding

  • Peptide competition assay using the phosphorylated peptide immunogen to confirm specificity

Essential Controls for ELISA:

• Standard curve: Using purified phosphorylated BCL2 protein or phospho-peptide at known concentrations.

• Blank wells: Containing all reagents except the primary antibody.

• Non-phosphorylated control: Samples containing BCL2 protein that is not phosphorylated at T74.

• Cross-reactivity controls: If available, include samples with BCL2 phosphorylated at other sites (S70, T56, S87) to confirm site-specificity.

Including these controls helps validate the specificity of staining or signal and ensures that experimental results accurately reflect the phosphorylation status of BCL2 at the T74 position.

How can researchers induce and verify T74 phosphorylation in experimental systems?

Inducing and verifying T74 phosphorylation of BCL2 in experimental systems requires careful selection of stimuli and validation methods:

Methods to Induce T74 Phosphorylation:

• Microtubule-targeting drugs: Paclitaxel and colchicine have been shown to induce multi-site phosphorylation of BCL2, including at T74 . Typical treatment concentrations range from 0.1-1 μM for 12-24 hours.

• JNK pathway activators: Anisomycin, UV radiation, or expression of constitutively active MEKK1 can activate JNK, potentially leading to BCL2 phosphorylation.

• ERK pathway stimulation: Growth factors like EGF or phorbol esters such as PMA can activate the ERK pathway, which may contribute to BCL2 phosphorylation .

• Cellular stress: Various forms of cellular stress, including oxidative stress and DNA damage, may trigger signaling cascades leading to BCL2 phosphorylation.

Verification Methods:

• Western blotting: Using the Phospho-BCL2 (T74) Antibody to detect the phosphorylated form, with total BCL2 antibody on parallel samples to normalize for expression levels.

• ELISA: Quantitative measurement of phosphorylated BCL2 levels using the antibody at 1:10000 dilution .

• Phosphorylation-specific mobility shift: Multi-site phosphorylated BCL2 often exhibits a characteristic mobility shift on SDS-PAGE.

• Mass spectrometry: For definitive confirmation of phosphorylation at T74 and determination of other concurrent phosphorylation events.

• In vitro kinase assay: Using synthetic peptides containing the T74 site (as shown in Table 1 from source ) to assess direct phosphorylation by purified kinases like JNK or ERK2.

Combining induction methods with multiple verification techniques provides the most robust evidence for T74 phosphorylation in experimental systems.

How does T74 phosphorylation affect BCL2's protein-protein interactions and conformational structure?

Phosphorylation at T74 appears to influence BCL2's functional properties through alterations in protein-protein interactions and conformational changes:

These structural and interactive changes highlight why monitoring T74 phosphorylation status is important for understanding BCL2's dynamic role in regulating apoptosis under different cellular conditions.

What are the challenges in interpreting conflicting data about BCL2 phosphorylation and its functional effects?

Researchers face several significant challenges when interpreting data about BCL2 phosphorylation, particularly when findings appear contradictory:

• Context-dependent phosphorylation effects: The functional impact of BCL2 phosphorylation can vary dramatically depending on:

  • Cell type (lymphoid versus myeloid, primary versus transformed)

  • Growth conditions (cytokine-dependent versus autonomous growth)

  • Type of apoptotic stimulus (intrinsic versus extrinsic pathway activation)

  • Pattern of phosphorylation (single-site versus multi-site)

• Temporal dynamics: The timing of phosphorylation events matters—transient versus sustained phosphorylation may have opposite effects on BCL2 function. Since phosphorylation is a dynamic process involving both kinases and phosphatases, results can vary based on when measurements are taken .

• Technical considerations:

  • Antibody specificity issues—some phospho-specific antibodies may have cross-reactivity with other phosphorylation sites

  • Detection sensitivity—low-level phosphorylation may be missed in some assays

  • Sample preparation methods that may alter phosphorylation status

• Mechanistic complexity: Multiple upstream signaling pathways converge on BCL2 phosphorylation, making it difficult to isolate the effects of individual kinases or stimuli .

To resolve contradictions, researchers should:

• Compare experimental methodologies carefully

• Consider the full context of each study

• Design experiments that systematically address variables like cell type, stimulus, and temporal dynamics

• Use multiple, complementary methods to detect and verify phosphorylation status

• Employ site-specific mutants (e.g., T74A) to definitively establish the contribution of specific phosphorylation sites

Understanding these nuances helps explain why BCL2 expression alone doesn't consistently correlate with patient outcomes or therapeutic resistance in clinical contexts .

How might understanding T74 phosphorylation contribute to developing targeted cancer therapies?

The phosphorylation status of BCL2 at T74 has important implications for cancer therapy development:

• Biomarker potential: T74 phosphorylation status could serve as a biomarker for:

  • Predicting response to microtubule-targeting drugs like paclitaxel, which induce multi-site BCL2 phosphorylation

  • Identifying tumors likely to respond to BH3 mimetic drugs, which target BCL2's anti-apoptotic function

  • Stratifying patients with follicular lymphoma or other BCL2-overexpressing cancers for appropriate therapy selection

• Drug development strategies:

  • Compounds that promote T74 phosphorylation (particularly as part of multi-site phosphorylation) might reduce BCL2's anti-apoptotic activity, potentially overcoming resistance to conventional therapies

  • Drugs targeting the kinases responsible for T74 phosphorylation could modulate BCL2 activity in a more nuanced way than direct BCL2 inhibitors

  • Combination therapy approaches that simultaneously target BCL2 expression and phosphorylation status could provide synergistic effects

• Resistance mechanism insights: Understanding how phosphorylation at T74 affects binding to pro-apoptotic partners and BH3 mimetic drugs could help explain why some patients develop resistance to BCL2-targeted therapies .

• Novel therapeutic target identification: The protein-protein interactions influenced by T74 phosphorylation, such as binding to Pin1, might represent novel therapeutic targets in BCL2-dependent cancers .

Despite these promising directions, translating knowledge about T74 phosphorylation into clinical applications requires further research to establish clear cause-effect relationships between specific phosphorylation patterns and therapeutic outcomes.

What are the most promising future research directions for understanding the role of T74 phosphorylation in BCL2 biology?

Several promising research directions could significantly advance our understanding of T74 phosphorylation in BCL2 biology:

• Structural biology approaches:

  • High-resolution structural studies comparing T74-phosphorylated versus non-phosphorylated BCL2 using cryo-EM or X-ray crystallography

  • NMR studies of the flexible loop domain under different phosphorylation conditions to map conformational changes more precisely

  • Molecular dynamics simulations integrating multiple phosphorylation sites to model complex phosphorylation patterns

• Single-cell analysis techniques:

  • Mass cytometry (CyTOF) to simultaneously measure multiple phosphorylation sites of BCL2 at the single-cell level

  • Live-cell imaging with phosphorylation-sensitive biosensors to track the dynamics of T74 phosphorylation in real-time

  • Single-cell RNA-seq combined with phospho-proteomics to correlate transcriptional states with BCL2 phosphorylation patterns

• Systems biology approaches:

  • Network analysis of kinase-phosphatase dynamics regulating BCL2 phosphorylation

  • Mathematical modeling of how multi-site phosphorylation, including at T74, affects the apoptotic threshold

  • Integration of phospho-proteomics with other omics data to understand contextual determinants of BCL2 phosphorylation

• Translational research directions:

  • Development of improved phospho-specific antibodies and detection methods for clinical samples

  • Correlation of T74 phosphorylation with response to various therapies in patient-derived xenograft models

  • Clinical trials incorporating T74 phosphorylation status as a biomarker for response to BCL2-targeting drugs

• Novel technology applications:

  • CRISPR-based approaches to introduce phospho-mimetic or phospho-dead mutations at T74

  • Optogenetic control of kinases to study temporal aspects of T74 phosphorylation

  • Proximity labeling techniques to identify proteins that specifically interact with T74-phosphorylated BCL2

These research directions would help address the current gaps in our understanding of how T74 phosphorylation contributes to BCL2's complex role in regulating apoptosis and its implications for disease states.