BRMS1L Antibody

What is BRMS1L and what are its primary cellular functions?

BRMS1L (Breast Cancer Metastasis-Suppressor 1-Like) is a 323 amino acid protein that localizes to the nucleus and functions as a component of the mSin3A/HDAC1 (histone deacetylase) complex. It participates in gene expression regulation through HDAC1-dependent transcriptional repression, which is vital for maintaining normal cellular functions and preventing uncontrolled cell growth . BRMS1L shares approximately 79% amino acid sequence similarity with breast cancer metastasis suppressor BRMS1, and was first isolated from the mSIN3A-HDAC transcriptional corepressor complex in 2004 . The primary function of BRMS1L appears to be suppressing target gene transcription through epigenetic mechanisms .

What molecular pathways does BRMS1L regulate in cancer cells?

BRMS1L regulates several crucial signaling pathways in cancer cells. In breast cancer, BRMS1L inhibits epithelial-mesenchymal transition (EMT) by epigenetically silencing FZD10, a receptor for Wnt signaling, through HDAC1 recruitment and histone H3K9 deacetylation at the promoter . This silencing inhibits aberrant activation of the WNT3-FZD10-β-catenin signaling pathway . In NSCLC, BRMS1L functions through a different mechanism, transcriptionally inhibiting glutathione peroxidase 2 (GPX2) expression, which consequently disrupts glutathione metabolism and increases reactive oxygen species (ROS) levels, ultimately inducing oxidative stress injury and apoptosis . Additionally, BRMS1L has been identified as a potential downstream mediator of the p53 pathway in cancer cell metastasis inhibition .

What criteria should researchers consider when selecting a BRMS1L antibody for specific applications?

When selecting a BRMS1L antibody, researchers should consider several critical factors based on their experimental objectives. First, evaluate the binding specificity and the exact epitope targeted by the antibody. For instance, some antibodies target the N-terminal region (AA 1-30), while others may target internal or C-terminal regions . Second, assess the host species and clonality of the antibody (polyclonal versus monoclonal), which impacts specificity and consistency between experiments . Third, confirm the validated applications for which the antibody has been tested, such as Western blotting (WB), immunoprecipitation (IP), immunofluorescence (IF), enzyme-linked immunosorbent assay (ELISA), or immunohistochemistry (IHC) . Fourth, verify species reactivity to ensure compatibility with your experimental model (human, mouse, rat) . Finally, consider whether conjugated forms (HRP, FITC, PE, etc.) might be beneficial for your particular experimental design .

How should researchers validate a new BRMS1L antibody before using it in critical experiments?

Validating a new BRMS1L antibody requires a systematic approach to ensure reliable results. Begin with positive and negative controls: use cell lines or tissues known to express high levels of BRMS1L (such as normal breast tissue) versus those with low expression (metastatic breast cancer cells) . Perform antibody titration experiments to determine optimal working concentrations for each application. For Western blotting validation, confirm that the detected band matches the expected molecular weight of BRMS1L (approximately 35-40 kDa). Validate specificity using BRMS1L knockout or knockdown cells in parallel with overexpression systems. For immunohistochemistry or immunofluorescence, include secondary antibody-only controls to assess background staining. Cross-validate results using at least two different antibodies targeting distinct epitopes of BRMS1L when possible. For definitive validation, perform an immunoprecipitation followed by mass spectrometry to confirm the identity of the captured protein. Document all validation steps thoroughly for reproducibility and method standardization in subsequent experiments.

What are the most common technical challenges when working with BRMS1L antibodies?

Several technical challenges commonly arise when working with BRMS1L antibodies. First, potential cross-reactivity with the highly homologous BRMS1 (breast cancer metastasis suppressor 1) protein, which shares 79% amino acid sequence similarity with BRMS1L, can lead to false-positive results . Second, low endogenous expression levels in certain cell types or tissues may necessitate optimization of protein extraction methods, loading higher protein amounts, or using signal enhancement techniques. Third, epitope masking can occur if BRMS1L is engaged in protein-protein interactions within the mSin3A/HDAC1 complex, potentially requiring optimization of sample preparation protocols. Fourth, phosphorylation or other post-translational modifications may affect antibody binding, requiring consideration of cell treatment conditions. Finally, subcellular localization challenges may arise, as BRMS1L primarily localizes to the nucleus, necessitating effective nuclear extraction protocols for accurate detection . Addressing these challenges requires careful optimization of experimental conditions specific to each application and thorough documentation of successful protocols.

What are the optimal protocols for detecting BRMS1L expression by Western blotting?

For optimal Western blotting detection of BRMS1L, researchers should follow this detailed protocol: Begin with efficient nuclear protein extraction since BRMS1L predominantly localizes to the nucleus as part of the mSin3A/HDAC1 complex . Use 4-12% gradient gels for optimal separation and resolution, loading 30-50 μg of nuclear protein extract per lane. For protein transfer, utilize PVDF membranes (0.45 μm pore size) and transfer at 30V overnight at 4°C to ensure complete transfer of the approximately 35-40 kDa BRMS1L protein. Block membranes with 5% non-fat dry milk in TBST for 1 hour at room temperature. For primary antibody incubation, use validated BRMS1L antibodies at optimized dilutions (typically 1:500 to 1:2000) in 5% BSA/TBST overnight at 4°C . After washing with TBST (3 × 10 minutes), incubate with appropriate HRP-conjugated secondary antibodies at 1:5000 dilution for 1 hour at room temperature. For enhanced sensitivity in detecting low BRMS1L expression, consider using chemiluminescent substrates with extended exposure times or employing signal enhancement systems. Include positive controls (normal breast tissue lysates) and negative controls (metastatic breast cancer cell lines with confirmed low BRMS1L expression) .

How can researchers effectively use BRMS1L antibodies for immunofluorescence and confocal microscopy?

For effective immunofluorescence and confocal microscopy with BRMS1L antibodies, researchers should implement the following protocol: Begin with appropriate fixation - 4% paraformaldehyde for 15-20 minutes works well for preserving nuclear proteins like BRMS1L. Permeabilize cells with 0.2% Triton X-100 in PBS for 10 minutes to allow antibody access to the nuclear-localized BRMS1L protein . Block non-specific binding with 5% normal serum (matched to secondary antibody host) containing 0.1% Triton X-100 for 1 hour at room temperature. For primary antibody incubation, use validated BRMS1L antibodies at optimized dilutions (typically 1:100 to 1:500) overnight at 4°C . After washing with PBS (3 × 5 minutes), incubate with fluorophore-conjugated secondary antibodies at 1:500 dilution for 1 hour at room temperature in the dark. Counterstain nuclei with DAPI (1 μg/ml) for 5 minutes. Use appropriate mounting medium containing anti-fade agents to preserve fluorescence. For confocal imaging, capture Z-stack images to ensure complete visualization of nuclear BRMS1L localization. For co-localization studies, combine BRMS1L antibodies with antibodies against other components of the mSin3A/HDAC1 complex to demonstrate functional associations . Implement quantitative image analysis of nuclear BRMS1L intensity across different cell types or experimental conditions for precise comparative studies.

What considerations are important when using BRMS1L antibodies for chromatin immunoprecipitation (ChIP) studies?

When conducting chromatin immunoprecipitation (ChIP) studies with BRMS1L antibodies, several critical considerations must be addressed. First, optimize crosslinking conditions given BRMS1L's role in the mSin3A/HDAC1 co-repressor complex - use 1% formaldehyde for 10 minutes at room temperature as a starting point . Select BRMS1L antibodies specifically validated for ChIP applications, as not all antibodies that work for Western blotting or immunofluorescence will perform adequately in ChIP . Thoroughly sonicate chromatin to fragments of 200-500 bp, verifying fragment size by gel electrophoresis. Include appropriate controls: IgG negative control, input control, and a positive control antibody against another component of the mSin3A/HDAC1 complex. For target selection, focus on promoters of known BRMS1L-regulated genes, such as FZD10 in breast cancer cells or GPX2 in NSCLC cells . Consider performing sequential ChIP (ChIP-reChIP) to analyze co-occupancy of BRMS1L with HDAC1 or other complex components to confirm functional complexes at specific genomic loci . Design primers for qPCR analysis of BRMS1L binding regions containing predicted transcription factor binding sites. For genome-wide analyses, combine ChIP with high-throughput sequencing (ChIP-seq) to identify novel BRMS1L target genes beyond the currently known FZD10 and GPX2 .

How does BRMS1L mechanistically inhibit breast cancer metastasis?

BRMS1L inhibits breast cancer metastasis through a sophisticated epigenetic mechanism targeting the Wnt signaling pathway. Specifically, BRMS1L recruits HDAC1 to the promoter of FZD10, a receptor for Wnt signaling, resulting in histone H3K9 deacetylation and subsequent epigenetic silencing of FZD10 expression . This silencing effectively inhibits the aberrant activation of the WNT3-FZD10-β-catenin signaling pathway that would otherwise promote cancer cell migration and invasion . Through this pathway inhibition, BRMS1L suppresses epithelial-mesenchymal transition (EMT), a critical process in cancer metastasis where epithelial cells lose their cell-cell adhesion properties and gain migratory and invasive characteristics . Additionally, BRMS1L is itself regulated post-transcriptionally by miR-106b, with increased miR-106b expression leading to reduced BRMS1L levels in breast cancer cells . This regulatory cascade creates a complex network controlling metastatic potential. In experimental models, RNA interference-mediated silencing of BRMS1L expression promotes metastasis of breast cancer xenografts in immunocompromised mice, whereas ectopic BRMS1L expression inhibits metastasis, providing direct evidence for its metastasis-suppressive function .

What role does BRMS1L play in non-small cell lung cancer (NSCLC) progression?

In non-small cell lung cancer (NSCLC), BRMS1L functions as a critical tumor suppressor through mechanisms distinct from its role in breast cancer. BRMS1L inhibits NSCLC proliferation and metastasis by transcriptionally suppressing glutathione peroxidase 2 (GPX2) expression . This suppression disrupts glutathione metabolism and increases reactive oxygen species (ROS) levels in cancer cells, inducing oxidative stress injury and ultimately leading to apoptosis . Mechanistically, overexpression of BRMS1L results in downregulation of GPX2 expression, while overexpression of GPX2 can rescue the growth disadvantage and oxidative stress caused by BRMS1L overexpression . Clinical analyses reveal that BRMS1L expression gradually decreases with increasing NSCLC stage according to the eighth edition of guidelines published by the International Association for the Study of Lung Cancer (IASLC) . Public database analyses show that low BRMS1L expression correlates with poor prognosis in NSCLC patients, while patients with high BRMS1L expression demonstrate longer survival . Interestingly, low BRMS1L expression in NSCLC cells causes relatively high levels of antioxidant accumulation to maintain cellular redox balance, potentially rendering these cancer cells more sensitive to treatment with ROS inducers like piperlongumine, suggesting therapeutic implications .

How can researchers effectively conduct BRMS1L knockdown and overexpression studies?

For effective BRMS1L knockdown and overexpression studies, researchers should implement the following comprehensive approach: For knockdown experiments, utilize both transient siRNA transfection and stable shRNA lentiviral transduction methods. Design at least three different siRNA or shRNA sequences targeting different regions of BRMS1L mRNA, and validate knockdown efficiency by both qRT-PCR and Western blotting . For overexpression studies, clone the full-length human BRMS1L cDNA (966 bp encoding 323 amino acids) into appropriate expression vectors with either constitutive (CMV) or inducible (Tet-On) promoters . Consider adding epitope tags (FLAG, HA, etc.) for enhanced detection and immunoprecipitation capabilities, but validate that these tags don't interfere with BRMS1L function. For both approaches, establish stable cell lines using appropriate selection markers to enable long-term studies of BRMS1L functions . Validate functional consequences of altered BRMS1L expression through multiple assays: proliferation (MTT, BrdU incorporation), apoptosis (Annexin V/PI staining, caspase activity), migration (wound healing, transwell), invasion (Matrigel-coated transwell), and ROS levels (DCFDA staining) . Perform rescue experiments where BRMS1L is re-expressed in knockdown cells or where downstream targets (FZD10 in breast cancer, GPX2 in NSCLC) are manipulated to confirm mechanistic relationships . Finally, extend in vitro findings to in vivo xenograft models to assess effects on tumor growth and metastasis formation .

What techniques can detect associations between BRMS1L and other components of the mSin3A/HDAC1 complex?

To detect associations between BRMS1L and other components of the mSin3A/HDAC1 complex, researchers should employ multiple complementary techniques. Co-immunoprecipitation (Co-IP) serves as the gold standard: use BRMS1L antibodies to immunoprecipitate the protein complex from nuclear extracts, then probe for associated proteins (mSin3A, HDAC1, etc.) by Western blotting, or vice versa . For endogenous protein interaction detection, proximity ligation assay (PLA) provides single-molecule resolution of protein-protein interactions within cells, allowing visualization and quantification of BRMS1L associations with complex components in situ. Implement bimolecular fluorescence complementation (BiFC) by fusing BRMS1L and potential interacting partners to complementary fragments of fluorescent proteins, which emit fluorescence when brought into proximity by protein interaction. For structural studies, perform size-exclusion chromatography followed by Western blotting to demonstrate co-elution of BRMS1L with mSin3A/HDAC1 complex components. Blue native PAGE can preserve native protein complexes for subsequent identification of components by Western blotting or mass spectrometry. Chromatin immunoprecipitation followed by re-ChIP (ChIP-reChIP) can demonstrate co-occupancy of BRMS1L and other complex components at specific genomic loci, such as the FZD10 promoter in breast cancer cells . Finally, employ mass spectrometry-based approaches like BioID or APEX proximity labeling to identify novel BRMS1L-interacting proteins, potentially expanding our understanding of the complete interactome.

How might BRMS1L status be leveraged for precision medicine approaches in cancer treatment?

BRMS1L status offers several promising avenues for precision medicine approaches in cancer treatment. First, BRMS1L expression levels could serve as a prognostic biomarker for patient stratification, as lower expression correlates with poorer outcomes in both breast cancer and NSCLC . Secondly, BRMS1L expression could function as a predictive biomarker for therapeutic response, particularly for treatments targeting pathways regulated by BRMS1L. For instance, in NSCLC, low BRMS1L expression correlates with cellular accumulation of antioxidants, potentially rendering these cells more sensitive to ROS-inducing agents like piperlongumine . This sensitivity has been demonstrated both in vitro and in vivo, suggesting a therapeutic vulnerability that could be exploited . Thirdly, researchers could explore epigenetic modifying drugs like HDAC inhibitors to modulate BRMS1L function or restore expression in cancers where it is suppressed. Additionally, targeting miR-106b, which negatively regulates BRMS1L expression, could represent another therapeutic strategy to restore BRMS1L levels in breast cancer . For tumors with low BRMS1L expression, implementing targeted inhibition of downstream effectors like FZD10/Wnt signaling in breast cancer or antioxidant pathways in NSCLC could provide synthetic lethality approaches . Finally, combination therapies that pair conventional chemotherapy with agents targeting BRMS1L-regulated pathways might enhance therapeutic efficacy while reducing required doses and associated toxicities.

What methodological approaches can resolve conflicting data on BRMS1L function across different cancer types?

Resolving conflicting data on BRMS1L function across different cancer types requires implementing several sophisticated methodological approaches. First, conduct systematic multi-omics analyses integrating transcriptomics, proteomics, and epigenomics data across cancer types to identify tissue-specific co-factors and regulatory networks that may explain differential BRMS1L functions . Second, develop isogenic cell line panels representing multiple cancer types modified to express equivalent levels of BRMS1L, allowing direct comparison of functional outcomes independent of expression level variations. Third, perform comprehensive ChIP-seq and ATAC-seq analyses across cancer types to identify tissue-specific BRMS1L binding sites and chromatin accessibility patterns that might explain differential target gene regulation . Fourth, utilize CRISPR/Cas9-mediated genome editing to introduce identical BRMS1L mutations across different cancer cell types, allowing assessment of mutation effects in diverse cellular contexts. Fifth, implement patient-derived organoid (PDO) and patient-derived xenograft (PDX) models across cancer types to evaluate BRMS1L function in more physiologically relevant systems than traditional cell lines. Sixth, conduct thorough analysis of BRMS1L interactome using BioID or IP-mass spectrometry across cancer types to identify differential protein-protein interactions that might explain context-specific functions . Finally, develop computational models incorporating these multi-dimensional datasets to predict cancer type-specific BRMS1L functions and generate testable hypotheses regarding apparent contradictions in experimental results.

How can single-cell technologies advance our understanding of BRMS1L's role in tumor heterogeneity?

Single-cell technologies offer powerful approaches to elucidate BRMS1L's role in tumor heterogeneity through several advanced methodologies. Single-cell RNA sequencing (scRNA-seq) can reveal cell subpopulations with differential BRMS1L expression within heterogeneous tumors, correlating expression with distinct cellular phenotypes and metastatic potential . Single-cell ATAC-seq can identify chromatin accessibility patterns associated with varying BRMS1L levels, providing insights into its epigenetic regulatory mechanisms across diverse cellular contexts. Single-cell ChIP-seq, although technically challenging, could map BRMS1L chromatin binding sites at the individual cell level, revealing potential cell-specific target genes and regulatory networks. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) can simultaneously analyze BRMS1L protein expression and transcriptional profiles at single-cell resolution, addressing potential discrepancies between mRNA and protein levels. For spatial context, technologies like multiplexed immunofluorescence, imaging mass cytometry, or spatial transcriptomics can map BRMS1L expression patterns within the tumor microenvironment, potentially revealing interactions with stromal or immune cells that influence its function. Single-cell multi-omics approaches combining genomic, transcriptomic, and epigenomic profiling could comprehensively characterize how genetic alterations influence BRMS1L expression and function within individual cells. Finally, lineage tracing combined with BRMS1L expression analysis could track the contribution of BRMS1L-expressing or BRMS1L-deficient cells to metastatic progression over time, directly addressing its role in clonal evolution during cancer progression .