Phospho-BCL2L1 (Ser62) Antibody

What is BCL2L1 and what role does phosphorylation at Ser62 play?

BCL2L1 (Bcl-xL) is a member of the BCL-2 protein family that functions as an anti-apoptotic regulator. BCL-2 family members form hetero- or homodimers and are involved in a wide variety of cellular activities . Phosphorylation of BCL2L1 at Ser62 has been detected in various cell lines treated with microtubule inhibitors including nocodazole, paclitaxel, vinblastine, vincristine, colchicine, and pironetin . This phosphorylation plays critical roles in:

• Mitotic progression during prometaphase, metaphase, and anaphase boundaries

• Spindle assembly and chromosome segregation

• DNA damage-induced G2 checkpoint

Notably, the function of phospho-BCL2L1(Ser62) in mitosis appears to be separable from its known anti-apoptotic function, as the Bcl-xL(Ser62Ala) phosphorylation mutant maintains its anti-apoptotic effect but shows different behavior during mitotic progression .

What types of Phospho-BCL2L1 (Ser62) antibodies are available for research?

Based on the search results, Phospho-BCL2L1 (Ser62) antibodies are predominantly available as:

Most commercial antibodies are developed against a phospho-specific peptide corresponding to residues surrounding S62 of human Bcl-XL, with the sequence being conserved in mouse and rat .

How should I optimize Western blot conditions for detecting phospho-BCL2L1(Ser62)?

For optimal Western blot results with phospho-BCL2L1(Ser62) antibodies:

• Sample preparation: Ensure phosphatase inhibitors are included in lysis buffers to prevent dephosphorylation during extraction.

• Protein amount: Load 20-40 μg of total protein per lane.

• Dilution optimization: Start with the recommended dilution range (1:500-1:2000) and optimize if needed.

• Detection: Use cell extracts from mitotic cells (e.g., nocodazole-treated cells collected by mitotic shake-off) as positive controls, as Bcl-xL is highly phosphorylated at Ser62 during early mitosis .

• Expected band: Look for a band at approximately 30 kDa, which is slightly higher than the calculated molecular weight (26 kDa) .

• Validation controls: Include samples treated with λ-phosphatase to confirm phospho-specificity, and consider including the Bcl-xL(Ser62Ala) mutant as a negative control if available .

What are the best fixation and staining protocols for immunofluorescence detection of phospho-BCL2L1(Ser62)?

For immunofluorescence applications:

• Fixation: Use 4% paraformaldehyde (15 minutes at room temperature) followed by permeabilization with 0.2% Triton X-100 (10 minutes).

• Blocking: Block with 5% normal serum from the same species as the secondary antibody for 1 hour.

• Primary antibody: Dilute phospho-BCL2L1(Ser62) antibody at 1:50-1:100 and incubate overnight at 4°C.

• Secondary antibody: Use fluorophore-conjugated anti-rabbit secondary antibody at recommended dilution (typically 1:200-1:500).

• Co-staining recommendations: For cell cycle studies, co-stain with:

  • γ-tubulin for centrosomes during early mitosis

  • Nucleolin for nucleolar localization during G2 arrest

  • PLK1, BubR1, or Mad2 for spindle-assembly checkpoint components

• Controls: Include a peptide competition assay using the phosphorylated peptide immunogen to confirm specificity.

How does phospho-BCL2L1(Ser62) status change during normal cell cycle progression?

During normal cell cycle progression, BCL2L1(Ser62) phosphorylation follows a dynamic pattern:

This dynamic phosphorylation pattern appears to be critical for proper mitotic progression, as expression of the phosphorylation mutant Bcl-xL(Ser62Ala) leads to various mitotic defects .

Which kinases are responsible for BCL2L1(Ser62) phosphorylation during mitosis versus DNA damage response?

The primary kinases responsible for BCL2L1(Ser62) phosphorylation differ depending on cellular context:

During normal mitosis:

• PLK1 (Polo-like kinase 1) is a major kinase responsible for Bcl-xL(Ser62) phosphorylation

• MAPK14/SAPKp38α also contributes significantly to this phosphorylation

• PLK1 inhibition abrogates Bcl-xL(Ser62) co-location with γ-tubulin in centrosomes

During DNA damage-induced G2 arrest:

• PLK1 remains a major kinase for Bcl-xL(Ser62) phosphorylation

• MAPK9/JNK2 becomes a significant contributor

• To a lesser extent, GSK3β is also involved

This context-dependent phosphorylation by different kinases likely represents distinct regulatory mechanisms for BCL2L1 function during normal mitosis versus DNA damage response.

How can I distinguish between the role of phospho-BCL2L1(Ser62) in mitosis versus its anti-apoptotic function?

To differentiate between these functions, implement the following strategies:

• Mutant expression: Compare cells expressing wild-type BCL2L1 versus the Ser62Ala mutant. Research demonstrates that Bcl-xL(Ser62Ala) retains anti-apoptotic activity but shows distinct behavior during mitotic progression .

• Cell cycle synchronization: Use double thymidine block to synchronize cells, then release and monitor through S, G2 and M phases while tracking phospho-BCL2L1(Ser62) levels.

• Specific inhibitors:

  • Treat cells with PLK1 or MAPK14/SAPKp38α inhibitors to prevent Ser62 phosphorylation without directly affecting apoptotic pathways

  • Test apoptotic response using standard apoptosis inducers with and without these kinase inhibitors

• Biochemical assays: Compare:

  • Binding to pro-apoptotic proteins (e.g., BAX, BAK) via co-immunoprecipitation

  • Interaction with mitotic regulators (e.g., Cdc20-Mad2-BubR1-Bub3 complexes)

• Microscopy approach: Track the different subcellular localizations:

  • Anti-apoptotic function: primarily at mitochondria

  • Mitotic function: at centrosomes with γ-tubulin and in mitotic cytosol with spindle-assembly checkpoint components

How does phospho-BCL2L1(Ser62) interact with the spindle assembly checkpoint (SAC) machinery?

Recent research has revealed important interactions between phospho-BCL2L1(Ser62) and SAC components:

• Protein interactions: In taxol- and nocodazole-exposed cells, phospho-Bcl-xL(Ser62) binds to Cdc20-Mad2-BubR1-Bub3-bound complexes, while the Bcl-xL(Ser62Ala) mutant does not .

• Co-localization: Phospho-BCL2L1(Ser62) co-localizes with PLK1, BubR1, and Mad2 in taxol-exposed cells .

• Functional impact: Silencing Bcl-xL expression or expressing the Bcl-xL(Ser62Ala) mutant leads to:

  • Increased frequency of cells with mitotic spindle defects including multipolar spindles

  • Chromosome lagging and bridging

  • Aneuploidy with micro-, bi-, or multi-nucleated cells

  • Failure to resolve mitosis within 6 hours

• Molecular mechanism: Phospho-Bcl-xL(Ser62) phosphorylation and dephosphorylation kinetics correlate with SAC/On and SAC/Off kinetics, suggesting a regulatory role in SAC signaling .

This interaction appears to be critical for proper chromosome segregation and maintaining chromosome stability during mitosis.

How does phospho-BCL2L1(Ser62) contribute to DNA damage-induced G2 checkpoint regulation?

During DNA damage response, phospho-BCL2L1(Ser62) plays a key role in G2 checkpoint regulation:

• Accumulation pattern: After DNA damage (e.g., etoposide treatment), phospho-Bcl-xL(Ser62) strongly accumulates in nucleolar structures during G2 arrest .

• G2 checkpoint stability: Cells expressing Bcl-xL(Ser62Ala) mutant are less stable at the G2 checkpoint and enter mitosis more rapidly than cells expressing wild-type Bcl-xL after DNA damage .

• Cdk1(cdc2) interaction: In nucleoli, phospho-Bcl-xL(Ser62) binds to and co-localizes with Cdk1(cdc2), the key cyclin-dependent kinase required for entry into mitosis .

• Mechanism: Phospho-Bcl-xL(Ser62) appears to stabilize G2 arrest by trapping Cdk1(cdc2) in nucleolar structures to slow mitotic entry .

• Kinase regulation: During DNA damage response, Bcl-xL(Ser62) phosphorylation is mediated primarily by PLK1 and MAPK9/JNK2, with GSK3β playing a minor role .

This function highlights how DNA damage affects the dynamic composition of the nucleolus and positions phospho-BCL2L1(Ser62) as an important component of the DNA damage response.

What are the differences in phospho-BCL2L1(Ser62) localization patterns between normal mitosis and DNA damage response?

Phospho-BCL2L1(Ser62) shows distinct localization patterns depending on cellular context:

The distinct localization patterns reflect different functions of phospho-BCL2L1(Ser62) in these contexts and provide valuable information for researchers studying its role in either normal cell cycle progression or DNA damage response.

What are common pitfalls when working with phospho-BCL2L1(Ser62) antibodies and how can they be avoided?

Common challenges and solutions when working with phospho-BCL2L1(Ser62) antibodies include:

• Loss of phosphorylation signal:

  • Always include phosphatase inhibitors in lysis buffers

  • Avoid repeated freeze-thaw cycles of samples

  • Process samples quickly and keep them cold

• High background in immunostaining:

  • Test multiple blocking solutions (BSA, normal serum, commercial blockers)

  • Optimize antibody concentration (typically 1:50-1:100 for IF/ICC)

  • Increase washing steps duration and volume

• Cell cycle-dependent detection issues:

  • Synchronize cells to enrich for phases with high phosphorylation

  • Use nocodazole treatment and mitotic shake-off to collect prometaphase/metaphase cells for positive controls

  • Remember that phospho-BCL2L1(Ser62) is dephosphorylated at telophase and cytokinesis

• Cross-reactivity concerns:

  • Validate specificity using Bcl-xL-depleted cells (siRNA)

  • Perform peptide competition assays

  • Include the Ser62Ala mutant as a negative control when possible

• Storage and stability issues:

  • Store aliquoted antibody at -20°C to avoid freeze-thaw cycles

  • Follow manufacturer recommendations for storage conditions (typically in 50% glycerol)

How can I optimize immunoprecipitation protocols to study phospho-BCL2L1(Ser62) interactions with mitotic regulators?

For successful immunoprecipitation of phospho-BCL2L1(Ser62) and its binding partners:

• Lysis buffer optimization:

  • Use mild NP-40 or CHAPS-based buffers to preserve protein-protein interactions

  • Include phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate)

  • Add protease inhibitors to prevent degradation

• Antibody selection:

  • For capturing phospho-BCL2L1(Ser62), use the specific phospho-antibody

  • For total BCL2L1 pulldown followed by phospho-detection, use well-validated total BCL2L1 antibodies

• Cell synchronization strategies:

  • For mitotic interactions: treat cells with nocodazole (0.35 μM, 4h) and collect by mitotic shake-off

  • For metaphase/anaphase boundary: release from nocodazole in the presence of MG-132 (25 μM)

  • For DNA damage response: treat with etoposide and collect at appropriate timepoints

• Subcellular fractionation:

  • For nucleolar interactions during DNA damage: perform enriched nucleolar extracts

  • For mitotic cytosolic interactions: prepare mitotic cytosolic fractions

• Validation approaches:

  • Perform reciprocal co-immunoprecipitations

  • Use phosphorylation mutants (Ser62Ala) as controls

  • Confirm specific interactions by mass spectrometry

Research has shown that phospho-BCL2L1(Ser62) interacts with different partners depending on cellular context, including Cdk1(cdc2) during DNA damage response and Cdc20-Mad2-BubR1-Bub3 complexes during spindle assembly checkpoint activation .

What are emerging techniques for studying BCL2L1(Ser62) phosphorylation dynamics in live cells?

Innovative approaches for monitoring BCL2L1(Ser62) phosphorylation dynamics include:

• Phospho-specific biosensors:

  • Design FRET-based biosensors incorporating BCL2L1 phosphorylation domains

  • Develop phospho-specific nanobodies tagged with fluorescent proteins

• Live-cell imaging with genetically encoded tags:

  • Express BCL2L1-FP fusions and correlate localization changes with cell cycle phases

  • Use split fluorescent protein complementation to visualize interactions with binding partners

• Optogenetic manipulation:

  • Develop light-inducible kinase systems to temporally control BCL2L1(Ser62) phosphorylation

  • Create optogenetic tools to disrupt phospho-BCL2L1(Ser62) interactions at specific subcellular locations

• Super-resolution microscopy:

  • Apply STED or STORM imaging to resolve phospho-BCL2L1(Ser62) localization at centrosomes and kinetochores

  • Combine with expansion microscopy for enhanced spatial resolution

• Single-cell analysis technologies:

  • Employ mass cytometry (CyTOF) with phospho-specific antibodies

  • Implement single-cell western blotting for phosphorylation analysis

These emerging techniques will enable researchers to gain deeper insights into the temporal and spatial regulation of BCL2L1(Ser62) phosphorylation during cell cycle progression and DNA damage response.

How might phospho-BCL2L1(Ser62) function be therapeutically targeted in cancer treatments?

The distinct role of phospho-BCL2L1(Ser62) in mitosis and DNA damage response suggests several therapeutic strategies:

• Combination therapy approaches:

  • Combine microtubule-targeting agents (taxanes) with inhibitors of PLK1 or MAPK14/SAPKp38α to prevent compensatory phosphorylation

  • Explore synergies between DNA-damaging agents and modulators of phospho-BCL2L1(Ser62) nucleolar accumulation

• Specific peptide inhibitors:

  • Design cell-penetrating peptides that mimic the Ser62 region to competitively inhibit kinase binding

  • Develop stapled peptides that disrupt interactions between phospho-BCL2L1(Ser62) and mitotic regulators

• Selective degradation strategies:

  • Create PROTACs (Proteolysis Targeting Chimeras) specific for phosphorylated BCL2L1

  • Explore phosphorylation-dependent molecular glues to induce degradation

• Targeted drug delivery systems:

  • Design nanoparticles containing kinase inhibitors that accumulate in mitotic cells

  • Develop antibody-drug conjugates targeting phospho-BCL2L1(Ser62) epitopes

• Cancer-specific applications:

  • Identify tumor types with aberrant BCL2L1(Ser62) phosphorylation patterns

  • Target chromosomally unstable cancers that may depend on phospho-BCL2L1(Ser62) for survival during abnormal mitosis