BCL2L13 Antibody

What is BCL2L13 and how does it function in cellular processes?

BCL2L13, also known as BCL-RAMBO or MIL1, is a member of the B-cell lymphoma 2 (BCL-2) protein family that plays essential roles in regulating apoptosis. It is a mitochondrially-localized protein with conserved BCL-2 homology motifs . The canonical human protein has 485 amino acid residues and a calculated molecular weight of 52.7 kDa, though it is often observed at approximately 85 kDa in experimental settings .

BCL2L13 has a unique structure containing all BH domains present in the BCL-2 protein family plus an additional BHNo domain comprising 250 amino acids at its C-terminal region . It functions primarily through two mechanisms:

• Apoptosis regulation: BCL2L13 can promote the activation of caspase-3 and facilitate apoptosis in various cellular contexts .

• Mitophagy mediation: It acts as a mammalian homolog of yeast Atg32, mediating mitophagy and mitochondrial fragmentation by binding to cleaved type II light chain 3 (LC3-II), allowing mitochondria to be engulfed within autophagosomes .

These dual functions position BCL2L13 as a critical regulator of cell fate decisions and mitochondrial quality control.

What is the tissue distribution and subcellular localization of BCL2L13?

BCL2L13 is widely expressed throughout the body, with particularly high expression levels in the heart, placenta, and pancreas . At the subcellular level, BCL2L13 is primarily localized to the mitochondria, consistent with its role in mitochondrial functions and apoptosis regulation . Some research also indicates nuclear localization , suggesting potential additional functions beyond its mitochondrial role.

In disease contexts, BCL2L13 shows altered expression patterns. It is highly expressed in acute myeloid leukemia (AML) cells and has been found to be overexpressed in glioblastoma (GBM) and other malignancies . This overexpression pattern suggests its potential involvement in therapy resistance mechanisms in cancer.

What are the recommended experimental conditions for using BCL2L13 antibodies in Western blot applications?

For Western blot applications, BCL2L13 antibodies should be used according to these recommended parameters:

When troubleshooting Western blots with BCL2L13 antibodies, researchers should note the significant difference between the calculated (53 kDa) and observed (85 kDa) molecular weights. This difference is likely due to post-translational modifications or the protein's unique structural properties. Begin with a middle-range dilution (1:3000) and adjust based on signal intensity and background levels.

It's essential to include positive controls such as HeLa cells or heart tissue lysates to validate antibody performance in each experimental run .

What protocols are recommended for immunohistochemistry with BCL2L13 antibodies?

For immunohistochemistry applications with BCL2L13 antibodies, follow these guidelines:

Effective antigen retrieval is critical for successful immunohistochemical detection of BCL2L13. The data suggests that TE buffer at pH 9.0 provides optimal epitope exposure, though citrate buffer at pH 6.0 can be used as an alternative . Always perform appropriate controls, including a negative control (omitting primary antibody) and a positive control using human pancreas cancer tissue, which has been validated for BCL2L13 detection .

For semi-quantitative analysis of BCL2L13 expression in tissue sections, establish a consistent scoring system based on staining intensity and percentage of positive cells to enable reliable comparisons between experimental groups.

How can BCL2L13's dual role in apoptosis and mitophagy be experimentally distinguished?

Differentiating between BCL2L13's functions in apoptosis versus mitophagy requires carefully designed experiments that selectively target each pathway:

For apoptosis studies:

• Measure caspase-3 activation levels following BCL2L13 manipulation

• Assess cytochrome c release from mitochondria

• Quantify PARP cleavage

• Use apoptosis-specific inhibitors (e.g., Z-VAD-FMK) to determine if effects are caspase-dependent

• Examine BCL2L13 interaction with other BCL-2 family proteins

For mitophagy studies:

• Use a dual-fluorescence reporter system (e.g., mito-mCherry-GFP) to visualize mitophagy

• Quantify the mitophagy/mitochondria ratio through image analysis

• Assess BCL2L13 colocalization with LC3-II

• Compare effects with known mitophagy inducers such as deferiprone (DFP)

• Examine Parkin recruitment and ubiquitination patterns

Research has shown that in BCL2L13 knockdown cells, the mitophagy/mitochondria scores were similar to control cells treated with the mitophagy inducer DFP, indicating that loss of BCL2L13 may enhance basal mitophagy levels despite its known role as a mitophagy receptor . This seeming contradiction highlights the importance of context-dependent analysis.

What is the role of BCL2L13 in metabolic programming and how does it impact cellular differentiation?

BCL2L13 plays a significant role in metabolic programming, particularly in the context of adipocyte differentiation and stromal cell fate determination:

• Oxidative phosphorylation regulation: BCL2L13 knockdown in 3T3-L1 cells significantly reduced oxygen consumption rate (OCR) during adipogenic differentiation .

• Glycolytic shift: Cells with BCL2L13 knockdown showed higher extracellular acidification rate (ECAR) and increased glycolytic ATP production, suggesting a metabolic reprogramming toward glycolysis .

• Adipogenesis promotion: BCL2L13 expression increases during adipogenic differentiation, and its knockdown significantly impairs lipid accumulation and reduces expression of adipogenic markers like PPARG .

This metabolic regulation appears to be critical for proper differentiation of mesenchymal stem cells. In bone marrow stromal cells from C3H mice, which have higher adipogenic potential, BCL2L13 expression was 2.7-fold higher than in cells from B6 mice during adipogenesis . The mechanism appears to involve mitochondrial quality control through mitophagy and protection against apoptosis during the differentiation process.

To study this function experimentally, researchers should:

• Monitor mitochondrial respiration using Seahorse XF analyzer during differentiation

• Track changes in mitochondrial mass and morphology

• Assess adipogenic marker expression (PPARG, FABP4, ADIPOQ)

• Evaluate the effects of BCL2L13 manipulation on cellular bioenergetics

• Examine how BCL2L13 levels correlate with differentiation capacity in different cell populations

What is BCL2L13's role in cancer pathophysiology and therapy resistance?

BCL2L13 has emerged as a significant factor in cancer biology, particularly in therapy resistance:

• In glioblastoma (GBM): BCL2L13 is overexpressed and functions as a ceramide synthase inhibitor. It binds to proapoptotic ceramide synthases 2 (CerS2) and 6 (CerS6), blocking their complex formation and activity . This inhibition prevents the generation of proapoptotic ceramide species, contributing to therapy resistance.

• In acute myeloid leukemia (AML): BCL2L13 is highly expressed and involved in preventing apoptosis . Research suggests it could be a potential treatment target for acute leukemia.

The relationship between BCL2L13 levels and ceramide synthase activity is inversely correlated in GBM tumors, providing a molecular explanation for the low levels of proapoptotic ceramide species in high-grade gliomas associated with poor survival .

To investigate BCL2L13's role in cancer:

• Analyze BCL2L13 expression across tumor grades and correlate with patient outcomes

• Assess the impact of BCL2L13 knockdown on therapy-induced apoptosis

• Measure ceramide synthase activity following BCL2L13 manipulation

• Investigate the binding dynamics between BCL2L13 and ceramide synthases using co-immunoprecipitation

• Evaluate the potential of BCL2L13 inhibition as a sensitizing strategy for conventional cancer therapies

How should researchers verify BCL2L13 antibody specificity and validate knockdown experiments?

Ensuring antibody specificity and effective knockdown validation is crucial for reliable BCL2L13 research:

For antibody specificity validation:

• Compare staining patterns across multiple antibody clones targeting different epitopes

• Perform peptide competition assays to confirm specific binding

• Include positive control samples with known BCL2L13 expression (e.g., HeLa cells, heart tissue)

• Use knockout/knockdown samples as negative controls

• Verify the expected molecular weight (note that the observed weight of 85 kDa differs from calculated 53 kDa)

For knockdown validation:

• Assess reduction at both mRNA and protein levels, as seen in studies showing ~90% mRNA reduction and 50% protein reduction following BCL2L13 siRNA treatment

• Use multiple siRNA/shRNA sequences to confirm phenotype specificity

• Include scrambled/non-targeting controls

• Perform rescue experiments by re-expressing siRNA-resistant BCL2L13

• Monitor knockdown stability over the experimental timeframe

In published studies, effective BCL2L13 knockdown showed morphological differences between knockdown and control cells, with significant impairment of adipocyte differentiation as demonstrated by decreased Oil Red O staining . This indicates that phenotypic changes can serve as functional validation of effective knockdown.

What are the key considerations for immunoprecipitation studies with BCL2L13 antibodies?

Immunoprecipitation (IP) is valuable for studying BCL2L13 protein interactions, particularly with other apoptosis regulators or ceramide synthases. Key considerations include:

When investigating protein interactions:

• Use crosslinking approaches to capture transient interactions

• Consider performing IPs under different cellular conditions (e.g., apoptotic stimulus, differentiation)

• Validate interactions through multiple approaches (e.g., proximity ligation assay, FRET)

• For BCL2L13-ceramide synthase interactions, optimize lysis conditions to preserve membrane protein associations

To specifically study BCL2L13's interaction with ceramide synthases, researchers should consider using mild detergents and avoiding harsh lysis conditions that might disrupt membrane-associated protein complexes. Co-immunoprecipitation followed by activity assays can directly assess how BCL2L13 binding affects ceramide synthase function.

How can researchers investigate the therapeutic potential of targeting BCL2L13 in cancer?

Exploring BCL2L13 as a therapeutic target requires multifaceted approaches:

• Develop specific BCL2L13 inhibitors or blocking antibodies to disrupt its interaction with ceramide synthases

• Create cell line and patient-derived xenograft models with BCL2L13 manipulation to evaluate therapy responses

• Perform combination studies with established cancer therapies to identify synergistic effects

• Assess the relationship between BCL2L13 expression and therapy resistance in patient samples

• Investigate whether BCL2L13 inhibition can restore ceramide synthase activity and sensitize resistant cells to apoptosis

BCL2L13's established role in inhibiting ceramide synthases in glioblastoma suggests that targeting this interaction could overcome therapy resistance . Similarly, in acute myeloid leukemia, where BCL2L13 is highly expressed and involved in preventing apoptosis, targeting BCL2L13 might enhance the efficacy of current treatments .

A comprehensive approach would involve correlating BCL2L13 levels with patient outcomes across different cancer types and treatment regimens to identify the clinical contexts where BCL2L13-targeted therapies might be most beneficial.

What are the latest methods for studying BCL2L13's role in mitochondrial dynamics and quality control?

Advanced techniques for investigating BCL2L13's mitochondrial functions include:

• Live-cell imaging with mitochondrial reporters to track dynamics in real-time

• Mitophagy flux assays using mito-Keima or mt-mCherry-GFP constructs

• CRISPR-Cas9 genome editing to generate BCL2L13 knockout models

• Proximity labeling approaches (BioID, APEX) to identify the BCL2L13 interactome at mitochondria

• Super-resolution microscopy to visualize BCL2L13's distribution on mitochondrial membranes

• Seahorse XF analysis to measure the impact on mitochondrial respiration

Studies have shown that BCL2L13 knockdown cells display metabolic reprogramming, with decreased oxygen consumption rate during adipogenic differentiation and increased glycolytic ATP production . This suggests BCL2L13 plays a key role in determining the balance between oxidative phosphorylation and glycolysis.

Researchers should design experiments that can distinguish between BCL2L13's direct effects on mitochondrial function versus indirect effects through altered cell differentiation or stress responses. Combining functional readouts (respiration, membrane potential) with morphological assessment (fragmentation, distribution) provides a more complete picture of how BCL2L13 influences mitochondrial health and cellular metabolism.