RBMS1 Antibody

What is RBMS1 and what cellular functions does it perform?

RBMS1, also known as C2orf12, is a member of a small family of proteins that bind single-stranded DNA/RNA. It contains two sets of ribonucleoprotein consensus sequences (RNP-CS) with conserved motifs RNP1 and RNP2, which are required for DNA binding. RBMS1 is primarily located in the nucleus and is highly expressed in placenta, lung, and heart tissues .

RBMS1 functions include:

• DNA replication regulation

• Gene transcription modulation

• Cell cycle progression control

• Apoptosis regulation

• Binding specifically to the DNA sequence motif 5'-[AT]CT[AT][AT]T-3'

• Interaction with regions upstream of the MYC gene

These diverse functions make RBMS1 a protein of interest in multiple research contexts, particularly in cancer biology and cellular differentiation studies.

What applications are suitable for RBMS1 antibody detection?

RBMS1 antibodies can be employed in multiple experimental applications with different sensitivities and specificities. Based on the available data, RBMS1 antibodies (such as the 83623-5-RR antibody) have been validated for the following applications:

It is essential to titrate the antibody in each testing system to obtain optimal results, as the optimal dilution may be sample-dependent . For detection by Western blot, researchers should expect a band at approximately 45 kDa, which corresponds to the observed molecular weight of RBMS1 .

What are the optimal conditions for RBMS1 antibody storage and handling?

Proper storage and handling of RBMS1 antibodies are crucial for maintaining their reactivity and specificity. The recommended storage conditions for RBMS1 antibodies (such as 83623-5-RR) are:

• Temperature: Store at -20°C

• Buffer: PBS with 0.02% sodium azide and 50% glycerol pH 7.3

• Stability: Stable for one year after shipment when stored properly

• Aliquoting: Not necessary for -20°C storage, though smaller volumes (20 μl) may contain 0.1% BSA

Researchers should avoid repeated freeze-thaw cycles as these can degrade antibody quality and reduce binding efficiency. When using the antibody, it should be thawed completely at room temperature and gently mixed before use. Working dilutions should be prepared fresh before each experiment for optimal results.

What positive and negative controls should be used with RBMS1 antibodies?

When designing experiments using RBMS1 antibodies, appropriate controls are essential for result interpretation:

Positive Controls:

• HeLa cells, HEK-293 cells, HepG2 cells, and DU145 cells have been validated for Western blot applications

• HepG2 cells have been validated for flow cytometry (intracellular) applications

• Placenta, lung, and heart tissue lysates (tissues with high RBMS1 expression)

Negative Controls:

• Secondary antibody-only control to assess non-specific binding

• RBMS1 knockdown cells (using validated shRNA constructs as described in literature)

• Isotype control (Rabbit IgG) to determine background signal

Including these controls helps ensure experimental validity and aids in troubleshooting if unexpected results occur.

How does RBMS1 contribute to cancer immunotherapy resistance?

Recent research has uncovered a significant role for RBMS1 in modulating cancer immunotherapy responses, particularly in triple-negative breast cancer (TNBC). RBMS1 is prevalent among immune-cold TNBC and functions as an immunosuppressive factor .

Mechanistically, RBMS1 regulates programmed death ligand 1 (PD-L1) expression through a novel pathway:

• RBMS1 stabilizes the mRNA of B4GALT1, a newly identified glycosyltransferase of PD-L1

• B4GALT1 promotes glycosylation of PD-L1, protecting it from ubiquitination and degradation

• When RBMS1 is depleted, B4GALT1 mRNA is destabilized, resulting in reduced PD-L1 glycosylation

• Reduced glycosylation promotes ubiquitination and subsequent degradation of PD-L1

• Decreased PD-L1 levels enhance cytotoxic T cell-mediated anti-tumor immunity

Importantly, the combination of RBMS1 depletion with CTLA4 immune checkpoint blockade or CAR-T treatment has shown enhanced anti-tumor T-cell immunity both in vitro and in vivo. This suggests that targeting RBMS1 could be a novel approach to improve immunotherapy efficacy in TNBC and potentially other cancers with similar mechanisms .

What methodologies are effective for RBMS1 knockdown experiments?

Based on the research data, successful RBMS1 knockdown has been achieved using shRNA-mediated approaches. When designing RBMS1 knockdown experiments, researchers should consider:

• Delivery System: Lentiviral transduction systems have been effectively used to deliver shRNA targeting RBMS1

• Selection Process: Puromycin selection has been used to isolate cells with stable RBMS1 knockdown

• Validation Methods: Western blotting with specific antibodies is recommended to confirm knockdown efficiency

• Controls: Scrambled control shRNA (scr ctrl) should be used as a comparison to distinguish specific effects of RBMS1 knockdown from non-specific effects of the knockdown procedure

• Phenotypic Assays: Various assays can be employed to assess functional consequences, including:

  • MTT assay for cell proliferation and viability

  • Oil Red O staining for lipid accumulation (in adipocyte differentiation studies)

  • RNA sequencing to identify differentially expressed genes

In adipogenic differentiation studies, RBMS1 knockdown resulted in differential expression of 430 genes compared to control cells, with 66% downregulated and 34% upregulated, suggesting a complex regulatory role .

How does RBMS1 regulate mRNA stability of target genes?

RBMS1 functions as an RNA-binding protein that can regulate the stability of target mRNAs, affecting their expression and subsequent protein levels. The research data reveals:

• Direct RNA Interaction: RBMS1 contains RNA recognition motifs (RRMs) that enable direct binding to target mRNAs

• B4GALT1 Regulation: RBMS1 has been shown to regulate the mRNA stability of B4GALT1, a glycosyltransferase involved in PD-L1 modification. Depletion of RBMS1 destabilizes B4GALT1 mRNA, leading to decreased protein expression

• Sequence-Specific Binding: RBMS1 binds specifically to DNA/RNA sequence motifs, with documented binding to the motif 5'-[AT]CT[AT][AT]T-3'

• Broader Transcriptomic Effects: RNA-seq analysis of RBMS1 knockdown cells revealed hundreds of differentially expressed genes, suggesting a wide regulatory network. In adipocyte studies, 430 genes showed altered expression upon RBMS1 knockdown

To investigate RBMS1-mediated mRNA stability, researchers can employ methodologies such as:

• RNA immunoprecipitation (RIP) to identify direct RNA binding targets

• Actinomycin D chase experiments to measure mRNA half-life in the presence or absence of RBMS1

• RNA-seq analysis following RBMS1 manipulation to identify global effects on gene expression patterns

Understanding these mechanisms provides insight into how RBMS1 functions in diverse cellular processes including cancer progression and metabolic regulation.

What is the relationship between RBMS1 and adipogenic differentiation?

Research into RBMS1's role in adipogenic differentiation has yielded interesting insights. Studies in 3T3-L1 cells, a model system for adipocyte differentiation, revealed:

• Expression Pattern: RBMS1 shows significant expression before and during the early stages of adipogenic differentiation of 3T3-L1 cells

• Functional Assessment: Despite this expression pattern, RBMS1 knockdown experiments showed that:

  • Cell proliferation and viability remained unaffected, as measured by MTT assay

  • Lipid droplet formation appeared similar to control cells

  • Quantification of oil droplets showed no difference in lipid content compared to scrambled control

• Transcriptomic Changes: Despite no obvious morphological differences, RNA sequencing revealed 430 differentially expressed genes in RBMS1 knockdown adipocytes compared to controls:

  • 66% of these genes were downregulated

  • 34% were upregulated

This suggests that while RBMS1 may not be essential for the gross morphological aspects of adipocyte differentiation, it plays a significant role in regulating the transcriptional program during this process. The specific pathways and biological processes affected by these gene expression changes would require further investigation through pathway enrichment analysis of the differentially expressed genes.

What are the optimal protocols for validating RBMS1 antibody specificity?

Validating antibody specificity is crucial for generating reliable research data. For RBMS1 antibodies, a comprehensive validation approach should include:

• Western Blot Analysis:

  • Confirm a single band at the expected molecular weight (45 kDa for RBMS1)

  • Compare signal across multiple cell lines known to express RBMS1 (HeLa, HEK-293, HepG2, DU145)

  • Include positive control lysates from tissues with high RBMS1 expression (placenta, lung, heart)

  • Test antibody in RBMS1 knockdown cells to confirm signal reduction

• Immunoprecipitation:

  • Perform IP followed by Western blot to confirm antibody pulls down RBMS1

  • Validate by mass spectrometry analysis of immunoprecipitated protein

• Immunofluorescence/Immunohistochemistry:

  • Confirm nuclear localization pattern consistent with RBMS1's known subcellular distribution

  • Compare staining pattern with multiple antibodies targeting different epitopes of RBMS1

• Flow Cytometry Validation:

  • Compare intracellular staining between positive control cells (e.g., HepG2) and cells with RBMS1 knockdown

  • Use appropriate isotype controls to determine background signal

• Cross-Reactivity Testing:

  • Test against recombinant proteins for RBMS1 paralogs (such as RBMS3) to ensure specificity

  • Evaluate potential cross-reactivity with similar RNA-binding proteins

These validation steps should be performed for each new lot of antibody and for each new application or cell type being studied.

What techniques are most effective for studying RBMS1-RNA interactions?

To investigate the RNA-binding properties of RBMS1 and identify its RNA targets, researchers should consider the following techniques:

• RNA Immunoprecipitation (RIP):

  • Use validated RBMS1 antibodies to pull down RBMS1-RNA complexes from cell lysates

  • Analyze bound RNAs by RT-qPCR for known targets or by RNA-seq for global analysis

  • Include appropriate controls (IgG control, input samples)

• Cross-Linking Immunoprecipitation (CLIP) and variations:

  • CLIP-seq or PAR-CLIP can be used to identify direct RNA binding sites with nucleotide resolution

  • UV cross-linking stabilizes protein-RNA interactions before immunoprecipitation

  • These techniques can map binding motifs across the transcriptome

• Electrophoretic Mobility Shift Assay (EMSA):

  • Use recombinant RBMS1 protein and labeled RNA probes

  • Test binding to the known sequence motif 5'-[AT]CT[AT][AT]T-3' and variants

  • Competition assays can determine binding specificity and affinity

• Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI):

  • Measure binding kinetics between purified RBMS1 and RNA oligos

  • Determine association and dissociation rates and binding affinities

• RNA Stability Assays:

  • Actinomycin D chase experiments in control vs. RBMS1-depleted cells

  • Measure half-lives of candidate target mRNAs (e.g., B4GALT1)

  • Luciferase reporter assays with 3'UTR elements of potential target RNAs

These techniques, used in combination, can provide comprehensive insight into the RNA-binding properties and regulatory functions of RBMS1 in various cellular contexts.

How should researchers troubleshoot non-specific binding with RBMS1 antibodies?

Non-specific binding is a common challenge when working with antibodies. For RBMS1 antibodies, consider the following troubleshooting approaches:

• Optimization of Blocking Conditions:

  • Test different blocking agents (BSA, milk, commercial blockers)

  • Extend blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Add 0.1-0.5% Tween-20 to washing buffers to reduce hydrophobic interactions

• Antibody Dilution Optimization:

  • Perform a dilution series across a wide range (1:5000-1:50000 for Western blot)

  • Find the minimum concentration that gives specific signal without background

  • Consider longer incubation times with more dilute antibody solutions

• Sample Preparation Improvements:

  • Ensure complete protein denaturation for Western blot (heat samples at 95°C for 5 minutes)

  • Use fresh samples and avoid protein degradation

  • For flow cytometry, optimize fixation and permeabilization protocols for intracellular staining

• Additional Controls:

  • Include RBMS1 knockdown samples as negative controls

  • Use peptide competition assays to confirm specific binding

  • Test multiple antibodies targeting different epitopes of RBMS1

• Buffer Optimization:

  • Adjust salt concentration to reduce ionic interactions

  • Add low concentrations of detergents (0.05-0.1% SDS) to reduce hydrophobic interactions

  • Consider adding protein competitors (1-5% BSA) to the antibody dilution

• Special Considerations for Flow Cytometry:

  • For intracellular staining, ensure complete permeabilization (0.25 μg per 10^6 cells in 100 μl suspension)

  • Include dead cell discrimination dyes

  • Use FcR blocking reagents to prevent Fc receptor binding

Systematic optimization of these parameters should help reduce non-specific binding while maintaining robust detection of RBMS1.

What experimental designs best elucidate RBMS1's role in cancer immunotherapy?

Based on current research, several experimental approaches can effectively investigate RBMS1's role in cancer immunotherapy:

• In Vitro Co-Culture Systems:

  • Co-culture RBMS1-manipulated cancer cells with activated T cells

  • Measure T cell activation markers, proliferation, and cytotoxicity

  • Assess cancer cell killing efficiency with or without RBMS1 depletion

  • Include PD-L1 blocking antibodies as positive controls

• RBMS1 Depletion Combined with Immunotherapy:

  • Test RBMS1 knockdown in combination with immune checkpoint inhibitors (anti-PD-1, anti-CTLA4)

  • Evaluate synergistic effects on cancer cell survival and T cell activation

  • Investigate combination with CAR-T therapy for enhanced anti-tumor effects

• Mechanistic Evaluation of PD-L1 Regulation:

  • Measure PD-L1 glycosylation status after RBMS1 manipulation

  • Assess B4GALT1 expression and activity in relation to RBMS1 levels

  • Investigate PD-L1 ubiquitination and degradation pathways

• In Vivo Models:

  • Establish syngeneic mouse models with RBMS1 knockdown tumors

  • Analyze tumor infiltrating lymphocytes (TILs) by flow cytometry

  • Measure tumor growth in response to combination therapy (RBMS1 depletion + immunotherapy)

  • Assess systemic immune responses and potential toxicities

• Clinical Correlation Studies:

  • Analyze RBMS1 expression in patient tumors in relation to:

    • PD-L1 levels

    • TIL abundance and activity

    • Response to immunotherapy

    • Patient outcomes and survival

These experimental approaches, particularly when used in combination, can provide comprehensive insights into RBMS1's role in cancer immunity and validate its potential as a therapeutic target for enhancing immunotherapy efficacy.