RBM6 Antibody

What is RBM6 and what are its key functional roles in cellular processes?

RBM6 (RNA Binding Motif Protein 6) is a nuclear protein that functions as an alternative splicing factor with established tumor suppressor activity. It contains two RNA recognition motifs (RRMs), one Zinc finger domain, a G-patch, and an OCRE domain, all of which contribute to its role in regulating alternative splicing . RBM6 localizes to nuclear foci that correspond to splicing speckles, consistent with its function in mRNA processing . Beyond splicing regulation, RBM6 has recently been identified as a novel regulator of homologous recombination (HR) repair of DNA double-strand breaks (DSBs), linking RNA processing with genomic stability maintenance . The protein specifically binds poly(G) RNA homopolymers in vitro, suggesting selective RNA interaction capabilities . RBM6 is also known by several alternative names including DEF3, Lung cancer antigen NY-LU-12, Protein G16, and RNA-binding protein DEF-3 .

What types of RBM6 antibodies are available for research applications and how should they be selected?

RBM6 antibodies are available in various formats targeting different epitopes of the protein. Researchers can choose from:

• C-terminal targeting antibodies, such as rabbit recombinant monoclonal antibodies suitable for Western blotting and immunocytochemistry/immunofluorescence

• Antibodies targeting specific amino acid regions (e.g., AA 1024-1123, AA 991-1040, AA 216-265, AA 530-770)

• N-terminal targeting antibodies with cross-reactivity to human and mouse samples

When selecting an RBM6 antibody, researchers should consider:

• The specific application (WB, IF, ICC, IHC)

• Species reactivity requirements

• The protein domain of interest

• Antibody format (monoclonal vs. polyclonal)

• Validation data for the specific application

For instance, the EPR15842 clone targets the C-terminal region and has been validated for Western blotting with a predicted band size of 129 kDa across multiple human cell lines including HUVEC, A549, HepG2, and HeLa .

How should RBM6 antibodies be optimized for Western blotting applications?

For optimal Western blotting results with RBM6 antibodies, researchers should consider the following protocol adaptations:

• Protein extraction: Use RIPA buffer (50 mM Tris pH 7.5, 1 M sodium chloride, 1% NP-40, 0.1% sodium deoxycholate, 1 mM EDTA) supplemented with complete protease inhibitor cocktail and RNase inhibitor for efficient extraction of nuclear proteins .

• Antibody dilution optimization: Start with manufacturer-recommended dilutions, which vary by antibody. For example:

  • Anti-RBM6 antibody [EPR15842] works effectively at 1/1000 dilution for HUVEC and A549 cell lysates

  • The same antibody can be used at 1/5000 dilution for HepG2 and HeLa cell lysates

• Secondary antibody selection: Use Goat Anti-Rabbit IgG (H+L), Peroxidase conjugated at 1/1000 dilution for optimal detection of rabbit primary antibodies .

• Protein loading: Load approximately 10 μg of total cell lysate per lane for clear visualization .

• Band size verification: Always verify that the observed band size matches the predicted molecular weight of 129 kDa for full-length RBM6 .

• Positive control selection: Include lysates from cells known to express RBM6 (such as A549, HeLa, or HepG2) as positive controls .

What are the recommended protocols for immunofluorescence staining of RBM6?

For immunofluorescence detection of RBM6, follow these methodological guidelines:

• Cell fixation: Fix cells with 4% paraformaldehyde to maintain cellular architecture and protein localization .

• Antibody dilution: For the EPR15842 antibody, use a 1/100 dilution for optimal staining in A549 cells .

• Secondary antibody: Use fluorophore-conjugated secondary antibodies such as Goat anti-rabbit IgG (Dylight® 488) .

• Expected localization pattern: RBM6 should appear primarily in nuclear foci corresponding to splicing speckles, consistent with its role in mRNA processing .

• Controls: Include RBM6-knockdown cells as negative controls to confirm antibody specificity.

What is the optimal protocol for immunohistochemical detection of RBM6 in tissue sections?

For effective immunohistochemical staining of RBM6 in formalin-fixed paraffin-embedded tissues:

• Tissue preparation: Fix tissues in 4% formalin followed by 70% ethanol processing .

• Deparaffinization and rehydration: Follow standard protocols to prepare tissue sections.

• Antigen retrieval: Perform antigen retrieval in 25 mM sodium citrate buffer pH 6.0 using a pressurized chamber for 2.5 minutes .

• Endogenous peroxidase blocking: Block with 3% H₂O₂ for 15 minutes .

• Non-specific binding reduction: Incubate sections with blocking solution (CAS Block) for 30 minutes .

• Primary antibody incubation: Apply RBM6 antibody and incubate overnight at 4°C .

• Secondary antibody application: Incubate with horseradish peroxidase-conjugated anti-rabbit immunoglobulin antibody for 30 minutes .

• Signal detection: Develop using 3,3-diamminobenzidine (DAB peroxidase kit) at room temperature .

• Counterstaining: Counterstain sections with hematoxylin to visualize cellular structures .

How does RBM6 contribute to DNA double-strand break repair mechanisms?

RBM6 plays a critical role in homologous recombination (HR) repair of DNA double-strand breaks through several mechanisms:

• Regulation of Fe65/APBB1 expression: RBM6 controls the alternative splicing-coupled nonstop-decay (AS-NSD) of Fe65, a positive regulator of HR repair. This represents a novel link between splicing regulation and DNA repair pathways .

• Impact of RBM6 deficiency: When RBM6 is knocked down, Fe65 protein levels are severely reduced, resulting in impaired HR repair. This defect can be rescued by restoring Fe65 expression, confirming the functional relationship between these proteins .

• Therapeutic implications: RBM6-deficient cells show increased vulnerability to ATM and PARP inhibitors, as well as pronounced sensitivity to cisplatin. This sensitivity is observed both in vitro and in mouse xenograft models using RBM6-deficient MDA-MB-231 breast cancer cells .

• Clinical relevance: The mechanistic understanding of RBM6's role in HR repair provides insight into why RBM6 mutations or deletions may contribute to genomic instability and cancer progression .

What is the significance of RBM6 expression patterns in cancer progression and metastasis?

RBM6 expression patterns have significant implications for cancer development and progression:

• Reduced expression in metastasis: Immunohistochemical analysis of human tissue microarrays has demonstrated that RBM6 protein levels are significantly reduced in metastatic breast tumors compared to primary tumors, suggesting a potential role in metastatic progression .

• Frequent genomic alterations: RBM6 is mapped to chromosome 3p21.3, a region frequently deleted in lung cancers associated with heavy smoking and in other tissue carcinomas .

• Mutation frequency: RBM6 is mutated in 2.4% of diverse human cancers across multiple origins (n=10,967 cases), including 1.5% of breast cancers (n=1,084 cases) and 3.1% of lung cancers (n=487 cases) .

• Tumor suppressor function: Multiple lines of evidence support RBM6 as a putative tumor suppressor gene, including its ability to repress growth and progression of laryngocarcinoma .

• Biomarker potential: RBM6 has been identified as a diagnostic biomarker for early detection of pancreatic cancer, extending its clinical relevance beyond breast and lung cancers .

How can RBM6 status be exploited for therapeutic targeting in cancer treatment?

The status of RBM6 in tumors offers several therapeutic opportunities:

• Synthetic lethality approaches: RBM6-deficient cancer cells show remarkable sensitivity to ATM and PARP inhibitors, suggesting potential targeted therapy approaches for tumors with low RBM6 expression .

• Cisplatin sensitivity: RBM6-deficient cancer cells exhibit pronounced sensitivity to cisplatin, both in vitro and in xenograft models. This indicates that conventional platinum-based chemotherapy may be particularly effective against RBM6-deficient tumors .

• Therapeutic stratification: Assessment of RBM6 status in tumors could potentially guide treatment decisions, particularly in advanced breast cancer where RBM6 loss is associated with metastasis .

• Restoration strategies: For tumors with reduced RBM6 expression, therapeutic approaches aimed at restoring Fe65 function might help overcome the HR repair deficiency .

• Combination therapies: The mechanistic understanding of how RBM6 deficiency affects DNA repair pathways opens possibilities for rational combination therapies targeting multiple aspects of the compromised repair machinery .

What are the recommended approaches for studying RBM6-RNA interactions?

To investigate RBM6-RNA interactions, researchers should consider these methodological approaches:

• RNA immunoprecipitation (RIP): Crosslink cells with 150 mJ/cm² at 254 nm, lyse in RIPA buffer supplemented with protease and RNase inhibitors, and immunoprecipitate using RBM6-specific antibodies. Process the samples with RQ1 DNaseI treatment according to the manufacturer's instructions .

• RNA binding studies: Leverage RBM6's known affinity for poly(G) RNA homopolymers in vitro as a positive control for binding experiments .

• Domain-specific analysis: Design experiments that distinguish between the functional contributions of RBM6's multiple RNA-binding domains (RRMs, Zinc finger, G-patch, and OCRE domains) .

• Splicing reporter assays: Utilize splicing reporter constructs to assess RBM6's impact on alternative splicing of specific target RNAs, particularly those involved in DNA repair pathways .

• Transcriptome analysis: Implement RNA-seq approaches to identify global changes in splicing patterns following RBM6 modulation, with particular attention to genes involved in homologous recombination repair .

What experimental approaches can distinguish between RBM6's roles in splicing versus other cellular functions?

To differentiate between RBM6's various cellular functions:

• Domain-specific mutations: Generate constructs with mutations in specific functional domains (RRMs, Zinc finger, G-patch, OCRE) to dissect which domains are essential for splicing versus DNA repair functions .

• Rescue experiments: In RBM6-knockdown cells, compare rescue with full-length RBM6 versus rescue with specific downstream effectors (e.g., Fe65/APBB1) to distinguish direct versus indirect effects .

• Nuclear fractionation: Perform biochemical fractionation to separate chromatin-associated, nucleoplasmic, and splicing speckle-associated pools of RBM6 to determine differential localization during various cellular processes .

• Temporal analysis: Assess RBM6 dynamics following DNA damage induction to determine whether its role in HR repair involves acute relocalization or primarily depends on its constitutive splicing functions .

• Interactome analysis: Compare RBM6 protein interaction partners in normal conditions versus genotoxic stress to identify context-specific interactions that may explain its multifunctional nature .

What are common issues with RBM6 antibodies and how can they be addressed?

When working with RBM6 antibodies, researchers may encounter several common issues:

• Multiple bands in Western blot:

  • Potential cause: Alternative splicing of RBM6 or proteolytic cleavage

  • Solution: Verify with multiple antibodies targeting different epitopes; include RBM6 knockdown controls; use fresh lysates with complete protease inhibitor cocktails

• Weak signal intensity:

  • Potential cause: Low expression levels or suboptimal antibody concentration

  • Solution: Increase antibody concentration; extend incubation time; optimize protein extraction method; use enhanced chemiluminescence detection systems

• Non-specific background:

  • Potential cause: Insufficient blocking or high antibody concentration

  • Solution: Optimize blocking conditions; titrate antibody concentration; include additional washing steps; pre-adsorb antibody with non-specific proteins

• Inconsistent results between applications:

  • Potential cause: Different epitope accessibility in various applications

  • Solution: Select antibodies validated for the specific application; consider using different antibodies for different applications

How can researchers validate the specificity of RBM6 antibodies?

To ensure RBM6 antibody specificity, implement these validation approaches:

• siRNA/shRNA knockdown controls: Compare staining patterns between control and RBM6-depleted samples to confirm signal specificity .

• Overexpression controls: In cells with low endogenous RBM6, introduce exogenous RBM6 expression and verify signal enhancement.

• Multiple antibody comparison: Use antibodies targeting different epitopes of RBM6 to confirm consistent detection patterns .

• Peptide competition: Pre-incubate antibody with the immunizing peptide to block specific binding and eliminate true signal.

• Band size verification: Confirm that the detected band matches the predicted molecular weight of 129 kDa for full-length RBM6 .

• Cross-reactivity assessment: Test antibody specificity across multiple species when working with non-human models, considering the availability of antibodies with validated reactivity against human, mouse, rat, cow, dog, rabbit, horse, bat, and monkey RBM6 .