RBM4 Antibody

What is RBM4 and what biological functions does it perform?

RBM4 (RNA binding motif protein 4) is a RNA-binding protein with a molecular weight of approximately 40 kDa. It contains two RRM (RNA Recognition Motif) domains that enable it to bind to specific RNA sequences . RBM4 plays critical roles in:

• Tumor suppression in various cancers including lung, colon, and gastric cancer

• Regulation of alternative splicing events that impact cell differentiation and development

• Inhibition of viral replication, particularly for viruses like Ebola virus

• Promotion of neuronal differentiation and neurite outgrowth through modulation of Numb isoform expression

• Enhancement of p53 mRNA stability, thereby activating the p53 signaling pathway

RBM4 primarily functions by binding to specific RNA sequences and influencing post-transcriptional regulation processes, which has significant implications for both normal cellular functions and disease states.

What are the recommended applications for RBM4 antibodies in research?

RBM4 antibodies have been validated for multiple research applications as evidenced by published literature. The following applications are well-established:

When selecting an application, consider your experimental goals and the validated reactivity of your antibody with your species of interest (human, mouse, and rat samples have demonstrated reactivity) .

What are the optimal dilution ratios for different RBM4 antibody applications?

Based on validated protocols, the following dilution ranges are recommended for RBM4 antibody (11614-1-AP):

It is strongly recommended to titrate the antibody in each testing system to determine optimal conditions for your specific experimental setup . Sample-dependent optimization is often necessary to achieve optimal signal-to-noise ratios.

How should I perform antigen retrieval for RBM4 immunohistochemistry?

For optimal RBM4 detection in fixed tissues using immunohistochemistry, antigen retrieval is crucial:

• Primary recommendation: Use TE buffer at pH 9.0 for heat-induced epitope retrieval (HIER)

• Alternative method: Citrate buffer at pH 6.0 can also be effective

Successful RBM4 detection has been reported in various tissues including:

• Human gliomas tissue

• Human lung cancer tissue

• Human stomach cancer tissue

The choice of antigen retrieval method may need to be empirically determined for each tissue type. In gastric cancer research, streptavidin-peroxidase (SP) staining technique after microwave-based antigen retrieval has been successfully employed .

How do I properly store and handle RBM4 antibodies to maintain reactivity?

To ensure optimal antibody performance and longevity:

• Storage temperature: Store at -20°C

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

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

• Aliquoting: Not necessary for -20°C storage for most preparations

• Special considerations: 20μl size preparations contain 0.1% BSA

For experimental use, allow antibody to equilibrate to room temperature before opening to prevent condensation that could introduce contaminants or promote degradation.

How can I use RBM4 antibodies to investigate its tumor suppressor functions?

RBM4 has demonstrated tumor suppressor properties in several cancer types. To investigate these functions:

• Expression analysis in tumor vs. normal tissues:

  • Use Western blot (1:500-1:2000 dilution) to quantify RBM4 expression

  • Perform IHC (1:50-1:500 dilution) on tissue microarrays to analyze expression patterns

  • Correlate expression with clinical parameters and survival data

• Functional studies:

  • Create RBM4-overexpression (RBM4-oe) cell lines using lentiviral vectors

  • Assess effects on proliferation using CCK-8 and EdU assays

  • Evaluate impact on cell cycle progression and apoptosis via flow cytometry

  • Examine migration capabilities through wound-healing assays

• Mechanistic investigation:

  • Explore pathway regulation using Gene Set Enrichment Analysis (GSEA)

  • Investigate p53 signaling pathway activation by measuring p53 mRNA stability

  • Examine EMT marker expression to understand impacts on migration/invasion

• In vivo verification:

  • Generate xenograft models using RBM4-overexpressing cells (typically 1×10^6 cells per injection)

  • Monitor tumor growth weekly (volume calculation: V=πAB^2/6)

  • Perform immunohistochemistry on harvested tumors to confirm RBM4 expression

Recent studies have shown that RBM4 inhibits clear cell renal cell carcinoma by enhancing p53 mRNA stability, providing a molecular mechanism for its tumor suppressive effects .

What are the best approaches to study RBM4's role in RNA splicing regulation?

To investigate RBM4's function in regulating alternative splicing:

• Target identification:

  • Perform RNA immunoprecipitation (RIP) using RBM4 antibodies to isolate bound RNA targets

  • Analyze RBM4 binding motifs and enriched sequences

  • Examine specific targets like Numb, where RBM4 has been shown to modulate alternative exon selection

• Splicing pattern analysis:

  • Use RT-PCR with exon-specific primers to detect alternative splice variants

  • Employ RNA-seq to identify global splicing changes upon RBM4 manipulation

  • Focus on neuronal differentiation models where RBM4 regulates Numb isoform expression

• Functional validation:

  • Create RBM4 knockdown and overexpression models

  • Examine expression of specific isoforms (e.g., Numb isoforms)

  • Correlate with downstream effects such as Mash1 expression in neuronal differentiation

• Domain-function analysis:

  • Generate RBM4 mutants lacking specific functional domains

  • The RRM1 domain has been identified as crucial for RNA binding, particularly to "CU" enrichment elements

  • Assess how domain mutations affect splicing patterns of target genes

When studying RBM4's splicing activity, it's important to consider cell type-specific effects, as splicing regulation may vary between tissues and developmental stages.

How can I optimize RNA immunoprecipitation (RIP) protocols using RBM4 antibodies?

For successful RIP experiments to identify RNA targets of RBM4:

• Sample preparation:

  • Crosslink cells with formaldehyde (typically 0.1-1%) to preserve RNA-protein interactions

  • Prepare cell lysates under conditions that maintain RNA integrity (use RNase inhibitors)

• Immunoprecipitation:

  • Use 0.5-4.0 μg of RBM4 antibody (11614-1-AP) per 1.0-3.0 mg of total protein lysate

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Include appropriate negative controls (IgG, non-target antibody)

  • Incubate overnight at 4°C with gentle rotation

• RNA recovery and analysis:

  • Reverse crosslinks and purify RNA using standard protocols

  • Analyze enriched RNAs via RT-qPCR for known targets or RNA-seq for discovery

  • Focus on regions containing "CU" enrichment elements, which have been identified as RBM4 binding sites

• Validation considerations:

  • Confirm RBM4 pulldown efficiency via Western blot

  • Validate select targets using reporter assays or functional studies

  • Consider CLIP-seq (Crosslinking and Immunoprecipitation sequencing) for greater resolution of binding sites

In viral research contexts, RIP has revealed that RBM4 binds directly to the PE1 and TSS regions within the 3'-leader of the Ebola virus genome, demonstrating its antiviral mechanism .

How can I investigate RBM4's role in antiviral responses, particularly against Ebola virus?

To study RBM4's antiviral functions:

• Expression analysis during infection:

  • Use Western blot (1:500-1:2000 dilution) to monitor RBM4 expression changes during viral infection

  • Compare expression levels between infected and uninfected cells

• Functional studies:

  • Manipulate RBM4 expression levels (overexpression or knockdown) in relevant cell lines

  • Assess impact on viral replication using plaque assays or viral RNA quantification

  • Research has shown that RBM4 inhibits Ebola virus replication in HEK293T and Huh-7 cells

• Mechanistic investigation:

  • Perform RIP assays to identify viral RNA sequences bound by RBM4

  • Focus on the 3'-leader region of the genomic RNA, particularly the PE1 and TSS regions containing "CU" enrichment elements

  • Examine the role of RBM4's RRM1 domain in the interaction with viral RNA

• Immune response analysis:

  • Measure cytokine expression in RBM4-manipulated cells during infection

  • RBM4 has been shown to upregulate expression of cytokines involved in host innate immune responses

• Protein domain analysis:

  • Create RBM4 mutants lacking specific domains

  • Assess which domains are essential for antiviral activity

  • The RRM1 domain is particularly important for binding to viral RNA

This research approach can provide insights into the potential of RBM4 as a novel target for antiviral strategies against Ebola virus and potentially other RNA viruses.

What experimental approaches can best elucidate RBM4's function in neuronal differentiation?

To investigate RBM4's role in neuronal development and differentiation:

• Expression profiling during differentiation:

  • Track RBM4 expression during neuronal differentiation using Western blot

  • Compare expression patterns in different neural cell types and developmental stages

• Alternative splicing analysis:

  • Focus on Numb isoform expression, as RBM4 modulates alternative exon selection of Numb

  • Use RT-PCR with isoform-specific primers to detect splice variants

  • Examine correlation between RBM4 levels and Numb isoform ratios

• Functional studies:

  • Manipulate RBM4 expression in neural progenitor cells or neuronal models

  • Assess effects on:

    • Neuronal marker expression (e.g., Mash1)

    • Neurite outgrowth and complexity

    • Neuronal differentiation timing and efficiency

• Morphological analysis:

  • Use immunofluorescence with neuronal markers to visualize morphological changes

  • Quantify neurite length, branching, and complexity in RBM4-manipulated cells

• Mechanistic investigation:

  • Examine the relationship between RBM4, Numb isoforms, and proneural gene expression

  • RBM4 has been shown to upregulate proneural Mash1 gene expression, possibly via specific Numb isoforms

  • Investigate downstream signaling pathways affected by RBM4-mediated splicing changes

These approaches can provide insights into how RBM4 contributes to neural development through its RNA processing functions, with potential implications for neurodevelopmental disorders and neural regeneration strategies.

What are common issues in Western blot detection of RBM4 and how can they be resolved?

When performing Western blot analysis with RBM4 antibodies, researchers may encounter several challenges:

• Weak or absent signal:

  • Increase antibody concentration (try 1:500 instead of 1:2000)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Increase protein loading (30-50 μg total protein)

  • Verify expression in your sample type (RBM4 is known to be expressed in brain, heart, and kidney tissues)

  • Use enhanced chemiluminescence (ECL) system for detection

• Multiple bands or non-specific binding:

  • Increase blocking time or blocking agent concentration

  • Optimize antibody dilution (start with 1:1000)

  • Include additional washing steps

  • RBM4 should appear at approximately 40 kDa (both calculated and observed)

• High background:

  • Use freshly prepared buffers

  • Increase washing duration and frequency

  • Dilute secondary antibody further

  • Ensure membrane is fully submerged during all incubations

• Inconsistent results between experiments:

  • Standardize protein extraction method

  • Use internal loading controls (β-actin is recommended)

  • Maintain consistent transfer conditions

  • Aliquot antibodies to avoid freeze-thaw cycles

For optimal RBM4 detection, using positive control samples like mouse brain tissue, human brain tissue, human heart tissue, human kidney tissue, or HeLa cells is recommended based on validated Western blot results .

How can I quantify RBM4 expression in IHC samples for reliable comparison across specimens?

For accurate quantification of RBM4 expression in immunohistochemistry:

• Standardized scoring system:

  • Implement a scoring system that accounts for both staining intensity and percentage of positive cells

  • Use a 4-grade intensity scale: 0 (no staining), 1 (light staining), 2 (moderate staining), and 3 (deep staining)

  • Calculate total score by multiplying the percentage of positive cells (0-100%) by the staining intensity score

• Technical considerations:

  • Process all samples using identical protocols (fixation, antigen retrieval, staining)

  • Include positive and negative controls in each batch

  • For RBM4, use TE buffer pH 9.0 for antigen retrieval (or alternatively citrate buffer pH 6.0)

  • Apply RBM4 antibody at consistent dilutions (1:50-1:500 recommended)

• Analysis approach:

  • Scan slides under low power (100×) to identify 5 areas with the highest RBM4 density (hot spots)

  • Photograph these areas at higher magnification (200×)

  • Have at least two experienced pathologists score independently using a double-blind design

  • Calculate inter-observer agreement (kappa statistic)

• Digital image analysis:

  • Consider using automated image analysis software for more objective quantification

  • Validate automated results against manual scoring

  • Maintain consistent imaging parameters across all specimens

This methodology has been successfully employed in cancer research to correlate RBM4 expression with clinical outcomes and tumor characteristics .

What controls should I include when performing functional studies with RBM4?

When conducting functional studies to investigate RBM4's biological roles, include these essential controls:

• Expression manipulation controls:

  • For overexpression: empty vector control with same promoter and selection marker

  • For knockdown: non-targeting siRNA/shRNA control

  • Validate expression changes via Western blot (1:500-1:2000 dilution)

• Functional assay controls:

  • Proliferation assays: Include positive control (growth factor) and negative control (serum starvation)

  • Cell cycle analysis: Use synchronized cell populations

  • Apoptosis assays: Include positive control (known apoptosis inducer)

  • Migration assays: Use both positive (chemoattractant) and negative (inhibitor) controls

• Mechanistic investigation controls:

  • For p53 pathway studies: Include p53 knockdown or inhibitor to confirm specificity

  • For RNA binding studies: Include mutated binding site controls

  • For splicing regulation: Examine multiple exons, including those not expected to be regulated by RBM4

• In vivo experiment controls:

  • For xenograft models: Inject control cells (left flank) and RBM4-manipulated cells (right flank) in the same animal for paired comparison

  • Include sham-operated controls

  • Validate protein expression in harvested tissues via Western blot and IHC

• Species considerations:

  • When using RBM4 antibodies across different model systems, ensure reactivity with the species being studied (validated in human, mouse, and rat)

How is RBM4 being investigated as a potential therapeutic target in cancer?

Current research into RBM4 as a therapeutic target in cancer focuses on several promising approaches:

• Restoration of RBM4 expression:

  • RBM4 is downregulated in multiple cancer types, including clear cell renal cell carcinoma, gastric cancer, and lung cancer

  • Approaches to restore expression include:

    • Viral vector-mediated gene delivery

    • Small molecules that enhance RBM4 transcription

    • Targeting upstream regulators of RBM4 expression

• Mechanism-based interventions:

  • Targeting the p53 pathway: RBM4 enhances p53 mRNA stability, activating p53 signaling and inhibiting cancer progression

  • Modulating specific splicing events regulated by RBM4 that contribute to cancer phenotypes

  • Influencing EMT (epithelial-mesenchymal transition) processes that RBM4 regulates to control migration and invasion

• Biomarker development:

  • RBM4 expression correlates with survival time in ccRCC patients

  • Potential use as prognostic or predictive biomarker

  • Development of standardized IHC protocols for clinical assessment

• Combination approaches:

  • Investigating synergy between RBM4-targeted therapies and conventional treatments

  • Exploring how RBM4 manipulation might enhance sensitivity to existing therapies

Research has demonstrated that RBM4 overexpression significantly reduces cancer cell proliferation, inhibits cell cycle progression, promotes apoptosis, and suppresses migration and invasion capabilities . These findings suggest that strategies to enhance RBM4 function could have therapeutic potential in multiple cancer types.

What is known about the interaction between RBM4 and viral replication, and how might this inform antiviral strategies?

RBM4's role in viral replication inhibition presents opportunities for novel antiviral approaches:

• Direct interaction with viral RNA:

  • RBM4 directly binds to the "CU" enrichment elements located in the PE1 and TSS of the 3'-leader region within the Ebola virus genome

  • This interaction occurs through RBM4's RRM1 domain

  • Binding inhibits viral mRNA production and subsequent viral replication

• Immunomodulatory functions:

  • RBM4 upregulates expression of cytokines involved in host innate immune responses

  • This creates a synergistic antiviral effect combining direct viral inhibition and enhanced immune activation

• Potential antiviral strategies:

  • Enhancing RBM4 expression or activity during viral infection

  • Developing peptide mimetics that replicate RBM4's binding to viral RNA

  • Screening for small molecules that strengthen RBM4-viral RNA interactions

  • Creating decoy RNA molecules that mimic viral binding sites to release RBM4 for antiviral activity

• Broader antiviral applications:

  • While current research focuses on Ebola virus, similar approaches might be effective against other RNA viruses

  • Investigation of RBM4's role in infections with other viruses is an emerging research area

The finding that RBM4 inhibits Ebola virus replication in both HEK293T and Huh-7 cells suggests that it might serve as a novel target for anti-EBOV strategy development . This research direction could contribute to addressing the significant public health challenges posed by viral outbreaks.

How do post-translational modifications affect RBM4 function and antibody detection?

Understanding RBM4's post-translational modifications is critical for both research applications and interpretation of results:

• Known modifications:

  • Phosphorylation: Affects RBM4's subcellular localization and splicing regulatory activity

  • Other potential modifications (methylation, ubiquitination) remain under investigation

• Impact on antibody detection:

  • Some modifications may mask epitopes recognized by certain antibodies

  • Consider using multiple antibodies targeting different regions when investigating modified forms

  • Phosphatase treatment prior to Western blot can help determine if bands represent phosphorylated forms

• Functional consequences:

  • Phosphorylation can alter RBM4's binding affinity for target RNAs

  • Modifications may regulate RBM4's interaction with other splicing factors

  • Changes in modification state during cell differentiation or stress response may redirect RBM4's splicing targets

• Experimental approaches:

  • Use phospho-specific antibodies when investigating specific modified forms

  • Consider 2D gel electrophoresis to separate differently modified forms

  • Mass spectrometry analysis can identify specific modification sites and types

  • Compare results across different cell states (stress, differentiation, disease) to understand regulatory patterns

This area remains underexplored and represents an important frontier in understanding the nuanced regulation of RBM4's diverse cellular functions.

What is the significance of RBM4 in neurodevelopmental disorders and neurological diseases?

Given RBM4's role in neuronal differentiation and neurite outgrowth, its potential significance in neurological conditions is an emerging area of investigation:

• Developmental roles:

  • RBM4 modulates alternative exon selection of Numb, a key regulator of neural development

  • It upregulates proneural Mash1 gene expression, influencing neuronal differentiation

  • These functions suggest potential involvement in neurodevelopmental disorders when dysregulated

• Splicing regulation in neurological contexts:

  • Many neurological diseases involve aberrant RNA splicing

  • RBM4's role as a splicing regulator positions it as a potential contributor to or therapeutic target for splicing-related neurological conditions

  • Investigation of RBM4 targets specific to neural tissues may reveal disease-relevant splicing events

• Research approaches:

  • Analysis of RBM4 expression in patient-derived samples from various neurological disorders

  • Examination of RBM4-regulated splicing patterns in disease models

  • Creation of neural-specific RBM4 knockout or transgenic models to assess developmental and functional impacts

  • Investigation of genetic variants affecting RBM4 expression or function in neurological disease cohorts

• Therapeutic implications:

  • Potential for RBM4-targeted approaches in disorders involving aberrant neural development

  • Possible applications in promoting neural regeneration after injury

  • Modulation of specific RBM4-regulated splicing events as a targeted therapeutic strategy

This represents a promising area for future research that could connect RBM4's known molecular functions to clinically relevant neurological conditions.

What experimental systems are most appropriate for studying different aspects of RBM4 function?

Selecting the optimal experimental system is crucial for investigating specific RBM4 functions:

• Cancer research:

  • Cell lines: Use established cancer cell lines with varying endogenous RBM4 expression

    • BGC823 cells have been successfully employed for RBM4 overexpression studies in gastric cancer

    • 786-O cells work well for renal cell carcinoma studies

  • Animal models: Xenograft models in immunodeficient nude mice have demonstrated RBM4's tumor-suppressive effects

  • Patient samples: IHC analysis of RBM4 in patient tumor samples provides clinical relevance

• Neuronal differentiation studies:

  • Neural stem/progenitor cells: Ideal for studying RBM4's role in differentiation decisions

  • Neuroblastoma cell lines: Can model neuronal differentiation in response to stimuli

  • Primary neuron cultures: Best for studying effects on neurite outgrowth and neuronal maturation

  • Brain slice cultures: Maintain native neural circuitry for more physiologically relevant studies

• Viral infection research:

  • HEK293T and Huh-7 cells: Validated for studying RBM4's effects on Ebola virus replication

  • BSL-4 facilities: Required for work with live Ebola virus

  • Viral replicon systems: Safer alternatives for mechanistic studies

• RNA binding and splicing mechanisms:

  • In vitro binding assays: For direct RNA-protein interaction studies

  • Minigene splicing reporters: To study regulation of specific alternative splicing events

  • CLIP-seq approaches: For genome-wide identification of binding sites

Each system offers distinct advantages, and the choice should be guided by the specific aspect of RBM4 biology being investigated and the available resources and expertise.

How can I design experiments to distinguish RBM4's direct effects from secondary consequences?

Establishing direct causality in RBM4 research requires careful experimental design:

• Temporal analysis:

  • Implement time-course experiments after RBM4 manipulation

  • Early changes (6-24 hours) are more likely to represent direct effects

  • Use inducible expression/knockdown systems for precise temporal control

• Domain-specific mutations:

  • Create RBM4 mutants lacking specific functional domains

  • The RRM1 domain is essential for RNA binding, particularly to "CU" enrichment elements

  • Compare effects of wild-type vs. binding-deficient mutants

• Direct binding validation:

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

  • Use CLIP-seq for higher-resolution mapping of binding sites

  • Validate binding with in vitro RNA-protein interaction assays

• Rescue experiments:

  • Knockdown endogenous RBM4 and rescue with wild-type or mutant versions

  • Differential rescue efficacy helps distinguish direct from indirect effects

  • Include expression-matched controls to account for dosage effects

• Immediate functional readouts:

  • For p53 pathway studies: Measure p53 mRNA stability directly after RBM4 manipulation

  • For splicing regulation: Examine pre-mRNA processing before secondary gene expression changes occur

  • For viral inhibition: Assess viral RNA binding and early replication steps

These approaches help establish clear mechanistic links between RBM4 and observed phenotypes, distinguishing its direct molecular functions from downstream consequences of altered gene expression or cellular state.

What techniques can I use to study the crosstalk between RBM4 and other RNA-binding proteins?

Investigating RBM4's interactions with other RNA-binding proteins requires specialized approaches:

• Protein-protein interaction studies:

  • Co-immunoprecipitation (Co-IP) using RBM4 antibodies (0.5-4.0 μg for 1.0-3.0 mg of total protein)

  • Proximity ligation assay (PLA) to visualize interactions in situ

  • FRET or BiFC for live-cell interaction studies

  • Mass spectrometry of RBM4-containing complexes to identify novel interacting partners

• Competitive binding analysis:

  • RNA electrophoretic mobility shift assays (EMSA) with purified proteins

  • Sequential or simultaneous RIP to examine shared targets

  • In vitro binding assays with labeled RNA and varying concentrations of competing proteins

• Functional interaction studies:

  • Combinatorial knockdown/overexpression of RBM4 and other RBPs

  • Analysis of splicing patterns when multiple RBPs are manipulated

  • Examination of post-translational modifications that may regulate interactions

• Computational approaches:

  • Comparison of binding motifs and target overlap between RBM4 and other RBPs

  • Network analysis of RBP interactions and cooperative/antagonistic relationships

  • Prediction of structural interaction interfaces

• Context-specific analysis:

  • Study interactions under different cellular conditions (stress, differentiation, disease)

  • Examine subcellular co-localization during different cellular processes

  • Investigate cell-type specific interaction patterns

These approaches can reveal how RBM4 functions within larger ribonucleoprotein complexes and regulatory networks, providing insight into cooperative and competitive relationships that modulate its activities in different cellular contexts.

How should I approach contradictory findings in RBM4 research literature?

When encountering conflicting reports about RBM4 functions or mechanisms: