Phospho-BCL2 (Ser70) Antibody

What is the biological significance of BCL2 phosphorylation at serine-70?

BCL2 phosphorylation at serine-70 (S70pBcl2) enhances the anti-apoptotic function of BCL2 by increasing its binding affinity to pro-apoptotic proteins such as BAX and BAD. This post-translational modification serves as a critical regulatory mechanism that determines BCL2's ability to protect cells from apoptosis. Studies have shown that S70pBcl2 functions as a redox sensor and modulator to prevent oxidative stress-induced DNA damage and cell death . In physiological contexts, this phosphorylation represents a dynamic process involving both kinases and phosphatases, allowing for rapid and reversible regulation of BCL2's activity and subsequent effects on cell viability .

How does S70 phosphorylation differ from other BCL2 phosphorylation sites?

While BCL2 can be phosphorylated at multiple sites including T69, S70, and S87, each site has distinct effects on BCL2 function. Specifically, phosphorylation at S70 maintains and enhances BCL2's anti-apoptotic effect, whereas phosphorylation at S87 inhibits its anti-apoptotic function . These site-specific modifications occur within the flexible loop domain (FLD), a 65-residue-long highly flexible region of BCL2 that plays a crucial role in regulating its activity. The differential effects of these phosphorylation sites highlight the complex regulatory mechanisms governing BCL2 function .

What is the relationship between S70pBcl2 and cellular redox state?

S70pBcl2 serves as a redox sensor that protects cells against oxidative stress-induced death. Research demonstrates that S70pBcl2 prevents oxidative stress-induced DNA damage by suppressing mitochondrial reactive oxygen species (ROS) production . Mechanistically, S70pBcl2 reduces the interaction between BCL2 and mitochondrial complex-IV subunit-5A, thereby reducing mitochondrial complex-IV activity, respiration, and subsequent ROS production. This redox-regulating function represents a novel facet of BCL2 biology that extends beyond its classical anti-apoptotic role and may contribute to the drug-resistant phenotypes observed in aggressive hematologic cancers .

What criteria should researchers consider when selecting a Phospho-BCL2 (Ser70) antibody?

When selecting a Phospho-BCL2 (Ser70) antibody, researchers should consider:

• Specificity: Verify that the antibody specifically recognizes the phosphorylated S70 epitope without cross-reactivity to non-phosphorylated BCL2 or other BCL2 family members. Most high-quality antibodies, such as the R.65.1 monoclonal antibody, are validated to ensure they are not cross-reactive with non-phosphorylated BCL2 at endogenous levels or with other BCL2 family members .

• Validated applications: Confirm that the antibody has been validated for your specific applications (WB, IHC, FC, etc.) with demonstrated reactivity in relevant species and cell types .

• Detection method compatibility: Ensure compatibility with your detection system and consider whether you need a monoclonal or polyclonal antibody based on your experimental needs.

• Published literature: Review publications that have successfully used the antibody in applications similar to yours.

The following table summarizes typical applications and dilutions for Phospho-BCL2 (Ser70) antibodies:

How can I validate the specificity of a Phospho-BCL2 (Ser70) antibody?

To validate the specificity of a Phospho-BCL2 (Ser70) antibody:

• Phosphatase treatment control: Treat cell lysates with lambda phosphatase prior to Western blotting. Loss of signal confirms specificity for the phosphorylated form.

• Phosphorylation-inducing treatments: Compare lysates from untreated cells with those treated with agents known to induce BCL2 phosphorylation, such as paclitaxel or calyculin A for Jurkat cells .

• Peptide competition assay: Pre-incubate the antibody with the phosphopeptide immunogen and observe signal reduction.

• Knockout/knockdown controls: Use BCL2-knockout cells or siRNA-mediated BCL2 knockdown as a negative control.

• Phosphomimetic mutants: Compare detection of wild-type BCL2 versus S70E (phosphomimetic) and S70A (phospho-null) mutants to confirm epitope specificity .

What are the optimal conditions for detecting S70pBcl2 by Western blotting?

For optimal detection of S70pBcl2 by Western blotting:

• Sample preparation:

  • Harvest cells rapidly and lyse immediately in buffer containing phosphatase inhibitors

  • For clinical samples, snap-freeze tissues immediately after collection

  • Avoid multiple freeze-thaw cycles

• Electrophoresis and transfer:

  • Use fresh reducing agents in sample buffer

  • Transfer to PVDF membrane rather than nitrocellulose for better signal

  • Maintain cold conditions during transfer

• Antibody incubation:

  • Use recommended dilutions (typically 1:2000-1:10000 for most commercial antibodies)

  • Incubate primary antibody overnight at 4°C

  • Consider 5% BSA instead of milk for blocking and antibody dilution to prevent phospho-epitope masking

• Positive controls:

  • Include lysates from paclitaxel-treated or calyculin A-treated Jurkat cells as positive controls

  • For research on oxidative stress, consider using cells with downregulated SOD1 or overexpressing Rac1 mutant (G12V)

• Expected results:

  • Phospho-BCL2 (Ser70) appears at approximately 26 kDa

How can I apply Phospho-BCL2 (Ser70) antibody in flow cytometry for studying apoptosis resistance?

For flow cytometry applications with Phospho-BCL2 (Ser70) antibody:

• Cell preparation:

  • Fix cells with 4% paraformaldehyde for 10-15 minutes

  • Permeabilize with 0.1% Triton X-100 or commercial permeabilization buffers

  • Block with 1-3% BSA to reduce non-specific binding

• Antibody staining:

  • Use approximately 0.06 μg antibody per 10^6 cells in 100 μl staining volume

  • Consider co-staining with apoptosis markers (e.g., Annexin V) and cell cycle markers

• Controls:

  • Include isotype controls to establish background

  • Use staurosporine-treated Jurkat cells as positive controls

  • Consider including S70A and S70E BCL2 mutant-expressing cells as reference points

• Dual analysis approach:

  • Combine with BH3 profiling to assess BCL2 functional status

  • For drug resistance studies, correlate S70pBcl2 levels with drug-induced apoptosis markers

• Data analysis:

  • Gate cells appropriately to exclude debris and aggregates

  • Consider median fluorescence intensity rather than percentage positive cells for quantitative analysis

What is the structural mechanism by which S70 phosphorylation enhances BCL2's anti-apoptotic function?

S70 phosphorylation enhances BCL2's anti-apoptotic function through conformational changes that increase its binding affinity for pro-apoptotic proteins. Molecular dynamics studies reveal that:

• Phosphorylation within the flexible loop domain (FLD) of BCL2 leads to a measurable reduction in structural flexibility . This reduced flexibility promotes better access of pro-apoptotic ligands to the binding groove of BCL2.

• In BCL2 with phosphomimetic mutations (T69E/S70E/S87E), new salt bridge formations occur, particularly between D196 and R63 with a hydrogen bond occupancy of 59%, compared to a maximum hydrogen bond occupancy of 29% between D196 and S87 in wild-type BCL2 .

• The addition of negative charges through phosphorylation creates favorable electrostatic interactions with positive charges elsewhere on the protein, stabilizing the structure and enhancing binding capacity .

• Surface plasmon resonance studies demonstrate that both phosphorylated BCL2 and the S70E phosphomimetic mutant exhibit enhanced binding to pro-apoptotic proteins Bim and Bak compared to unmodified BCL2 .

• Interestingly, the S70A mutant also shows enhanced binding to Bim and Bak, suggesting that the key mechanism may involve driving BCL2 to a more active conformation rather than simply providing a charge modification for specific phosphoepitope-directed interactions .

These structural changes explain mechanistically why S70pBcl2 provides greater protection against apoptosis induced by various chemotherapeutic agents.

How does S70pBcl2 contribute to cancer drug resistance mechanisms?

S70pBcl2 contributes to cancer drug resistance through multiple mechanisms:

Understanding these mechanisms has important implications for developing strategies to overcome drug resistance in cancer therapy.

What are the differences in experimental outcomes between using phosphomimetic (S70E) versus phospho-null (S70A) BCL2 mutants?

The literature shows complex and sometimes contradictory findings regarding S70E and S70A BCL2 mutants:

These findings suggest that:

• The simple model of S70E mimicking phosphorylation while S70A blocks it may be incomplete.

• Both mutations may alter BCL2 conformation in ways that enhance its anti-apoptotic function, but through potentially different mechanisms.

• The S70A mutant may lock BCL2 into a conformation that resembles the active state achieved through phosphorylation, despite lacking the phosphorylation itself .

• For experimental design, researchers should consider including both mutants alongside wild-type and endogenously phosphorylated BCL2 to fully understand the mechanism being studied.

These complexities highlight the need for careful interpretation of experimental results using these mutants.

How can I distinguish between single-site (S70) and multi-site BCL2 phosphorylation in my experiments?

Distinguishing between single-site S70 phosphorylation and multi-site phosphorylation requires strategic experimental approaches:

• Antibody selection:

  • Use site-specific antibodies like Phospho-BCL2 (Ser70) that do not cross-react with other phosphorylation sites

  • Compare with antibodies that recognize multiple phosphorylation sites (T69/S70/S87)

• Phosphorylation-specific Western blot patterns:

  • S70-only phosphorylation typically shows a single band at 26 kDa

  • Multi-site phosphorylation (particularly after paclitaxel treatment) often results in a mobility shift with multiple bands visible

• Mutant constructs:

  • Generate and compare single-site mutants (S70A or S70E) versus multi-site mutants (T69A/S70A/S87A or T69E/S70E/S87E)

  • Use these to establish phosphorylation-specific phenotypes

• Phospho-peptide mapping:

  • Perform mass spectrometry after immunoprecipitation to identify all phosphorylated residues

  • Use 2D phospho-peptide mapping to distinguish phosphorylation patterns

• Kinase inhibitor approach:

  • Different kinases preferentially phosphorylate different BCL2 sites

  • Use selective inhibitors of PKC (for S70) versus JNK (for T69/S87) to distinguish site-specific effects

What are the best experimental models for studying S70pBcl2's role in cancer drug resistance?

The optimal experimental models for studying S70pBcl2's role in cancer drug resistance include:

• Cell line models:

  • Hematologic cancer cell lines (Jurkat, K562) show robust BCL2 phosphorylation responses

  • Generate stable cell lines expressing wild-type, S70E, or S70A BCL2 for comparative studies

  • Use transient transfection systems with EGFP-tagged constructs for flow cytometry-based apoptosis assays

• Primary patient samples:

  • CLL and lymphoma patient-derived primary cells demonstrate clinically relevant S70pBcl2 responses

  • Correlation between S70pBcl2 levels and clinical outcomes provides translational relevance

• In vivo models:

  • BCL2-knockout mice with reconstitution of wild-type or mutant BCL2 (S70A/S70E)

  • Patient-derived xenograft models maintaining the phosphorylation status of the original tumor

• Induction conditions:

  • Paclitaxel treatment (50-100 nM, 16-24 hours) reliably induces BCL2 phosphorylation

  • Calyculin A (phosphatase inhibitor) rapidly increases phosphorylation levels

  • Oxidative stress induction using superoxide dismutase 1 (SOD1) downregulation

• Functional readouts:

  • Combine apoptosis assays with measurements of BCL2 binding to pro-apoptotic partners

  • Assess mitochondrial parameters (membrane potential, ROS production) alongside S70pBcl2 levels

  • Measure DNA damage (γH2AX) in relation to S70pBcl2 levels

How can I resolve contradictory results regarding S70pBcl2's function in my experimental system?

When facing contradictory results regarding S70pBcl2 function:

• Review experimental context:

  • Cell type specificity: The effect of S70pBcl2 may vary between hematopoietic cells versus solid tumor cells

  • Timing: Acute versus chronic phosphorylation may have different outcomes

  • Degree of phosphorylation: Partial versus complete phosphorylation may produce different results

• Consider multi-site phosphorylation:

  • S70 phosphorylation alone enhances anti-apoptotic function

  • Multi-site phosphorylation (T69/S70/S87) can have different effects than single-site phosphorylation

  • Use site-specific antibodies to distinguish these patterns

• Validate antibody specificity:

  • Confirm phospho-specificity using phosphatase treatments

  • Verify absence of cross-reactivity with other BCL2 family members

• Examine binding partners:

  • Different pro-apoptotic binding partners (BAK vs. BAX vs. BIM) may be affected differently

  • Perform co-immunoprecipitation studies to confirm actual binding changes

  • Consider using surface plasmon resonance to quantify binding affinities

• Cellular compartment analysis:

  • Examine S70pBcl2 localization (mitochondrial, ER, cytosolic)

  • Different pools of BCL2 may have distinct phosphorylation states and functions

• Validate with multiple techniques:

  • Combine biochemical, cellular, and structural approaches

  • If results differ between systems, identify the specific experimental variables responsible

What are the critical controls needed when studying the relationship between S70pBcl2 and oxidative stress?

When investigating S70pBcl2 and oxidative stress relationships, include these critical controls:

• Redox state verification:

  • Measure cellular ROS levels using multiple methods (DCF-DA, MitoSOX)

  • Include both general and mitochondria-specific ROS indicators

  • Verify that interventions actually change ROS levels as expected

• Phosphorylation status controls:

  • Compare untreated, ROS-inducer treated, and antioxidant-treated conditions

  • Include phosphatase-treated samples as negative controls

  • Use S70E and S70A mutants as comparative references

• Mitochondrial function assessment:

  • Measure mitochondrial complex-IV activity alongside S70pBcl2 levels

  • Assess BCL2 interaction with complex-IV subunit-5A with co-immunoprecipitation

  • Monitor mitochondrial membrane potential changes

• DNA damage correlation:

  • Quantify γH2AX foci as a measure of DNA damage

  • Use neutral and alkaline comet assays to distinguish single and double-strand breaks

  • Establish temporal relationship between phosphorylation, ROS, and DNA damage

• Intervention controls:

  • Use specific ROS scavengers (MitoTEMPO for mitochondrial ROS)

  • Apply site-specific phosphatase activators like FTY720

  • Include positive controls for different cell death pathways (apoptosis vs. necrosis)

These controls help distinguish correlation from causation in the complex relationship between S70pBcl2 and oxidative stress.