rnf152 Antibody

What is RNF152 and why is it important in cellular research?

RNF152 (ring finger protein 152) is a 203 amino acid protein characterized by a RING-type zinc finger motif that plays a crucial role in the ubiquitination pathway of protein degradation. This motif consists of a conserved cysteine-rich domain capable of binding two zinc ions, a feature essential for the protein's structural integrity and function . The ubiquitination process mediated by RNF152 regulates various cellular processes, including degradation of misfolded proteins and modulation of signaling pathways, thereby maintaining cellular homeostasis . The gene encoding RNF152 is located on human chromosome 18, a region associated with several genetic disorders, including Trisomy 18 and various cancers . Recent research has revealed RNF152's role in positively regulating TLR/IL-1R signaling pathways, highlighting its importance in inflammatory responses and immune function .

What types of RNF152 antibodies are available for research applications?

Currently, several monoclonal antibodies targeting RNF152 are available for research purposes. The most documented include:

These antibodies are available in both non-conjugated forms and various conjugated formats, including agarose, horseradish peroxidase (HRP), phycoerythrin (PE), fluorescein isothiocyanate (FITC), and multiple Alexa Fluor® conjugates, allowing researchers flexibility in experimental design .

What are the standard applications for RNF152 antibodies in basic research?

RNF152 antibodies can be utilized in multiple standard laboratory techniques, each providing different insights into RNF152 expression, localization, and function:

• Western blotting (WB): For detecting and quantifying RNF152 protein levels in cell or tissue lysates. This application is particularly useful for monitoring expression changes under different experimental conditions or treatments .

• Immunoprecipitation (IP): For isolating RNF152 protein complexes from cellular lysates to study protein-protein interactions, identifying binding partners that might be involved in RNF152's regulation or function .

• Immunofluorescence (IF): For visualizing the subcellular localization of RNF152, which can provide insights into its functional dynamics in different cellular compartments .

• Enzyme-linked immunosorbent assay (ELISA): For quantitative measurement of RNF152 levels in biological samples, useful for high-throughput screening applications .

When designing experiments with these applications, researchers should include appropriate positive and negative controls to ensure specificity and reliability of results.

How can RNF152 antibodies be optimized for studying its role in TLR/IL-1R signaling pathways?

To effectively investigate RNF152's role in TLR/IL-1R signaling pathways, researchers should consider the following methodological approaches:

• Co-immunoprecipitation with signaling components: Use RNF152 antibodies to perform co-IP experiments with components of the TLR/IL-1R signaling pathway, particularly MyD88, as research has shown that RNF152 facilitates MyD88 oligomerization . This approach can help elucidate the molecular mechanisms by which RNF152 positively regulates these signaling pathways.

• Proximity ligation assays: Combine RNF152 antibodies with antibodies against TLR/IL-1R pathway components to visualize and quantify protein-protein interactions in situ, providing spatial context for these interactions within the cell.

• Immunoblotting for pathway activation: Following stimulation with IL-1β or TLR ligands like LPS, use phospho-specific antibodies alongside RNF152 antibodies to monitor the activation status of downstream effectors such as IKKα/β and p38, which have been shown to be impaired upon RNF152 knockdown .

• Chromatin immunoprecipitation (ChIP): Combine RNF152 antibodies with ChIP to investigate whether RNF152 associates with chromatin regions controlling inflammatory gene expression, as RNF152 affects the transcription of inflammatory cytokines such as IL-1β, IL-6, and IL-8 .

Research has demonstrated that RNF152 deficiency impairs IL-1β-, LPS-, and PGN-induced transcription of inflammatory genes, indicating its requirement for IL-1R-, TLR2-, and TLR4-mediated signaling but not TLR3-mediated signaling . These methodological approaches can help further characterize the specificity and mechanisms of RNF152's involvement in these pathways.

What are the key considerations when using RNF152 antibodies to investigate its E3 ubiquitin ligase activity?

When investigating RNF152's E3 ubiquitin ligase activity, researchers should consider these methodological approaches:

• In vitro ubiquitination assays: Use immunoprecipitated RNF152 (using specific antibodies) to conduct in vitro ubiquitination assays with potential substrate proteins. This approach can help identify direct targets of RNF152's E3 ligase activity.

• Ubiquitination site mapping: Combine RNF152 antibody-based immunoprecipitation with mass spectrometry to identify ubiquitination sites on substrate proteins. This technique can reveal the specific lysine residues targeted by RNF152.

• Analysis of ubiquitin chain topology: Use antibodies specific for different ubiquitin linkages (K48, K63, etc.) alongside RNF152 antibodies to determine the type of polyubiquitin chains formed by RNF152. Research has indicated that RNF152 mediates K63-linked polyubiquitination of RagA in mTORC1 signaling .

• Domainx-based mutagenesis studies: When using RNF152 antibodies to study mutant forms of the protein, ensure the epitope recognized by the antibody is not affected by the mutations, particularly those involving the RING domain, which is critical for E3 ligase function.

The RING-type zinc finger motif of RNF152 is crucial for its E3 ubiquitin ligase activity . Interestingly, research has shown that RNF152 potentiates IL-1β- and LPS-induced NF-κB activation in an ubiquitination-independent manner , suggesting it has functions beyond its E3 ligase activity that should be considered when designing experiments.

How can researchers effectively use RNF152 antibodies to analyze its role in inflammatory responses in vivo?

To effectively study RNF152's role in inflammatory responses in vivo, researchers should implement these methodological approaches:

• Immunohistochemistry in tissue samples: Use RNF152 antibodies for immunohistochemical analysis of inflamed tissues from wild-type and RNF152-deficient mice to correlate RNF152 expression with inflammatory markers.

• Flow cytometry of immune cells: Employ conjugated RNF152 antibodies (such as PE or FITC conjugates) for flow cytometric analysis of immune cell populations to assess RNF152 expression levels in different immune cell types during inflammatory responses.

• Ex vivo analysis of primary cells: Isolate primary cells (MEFs, BMDMs, BMDCs) from wild-type and RNF152-deficient mice, stimulate with inflammatory mediators (IL-1β, LPS, PGN), and use RNF152 antibodies to correlate protein expression with inflammatory responses.

• Cytokine profiling in animal models: Use RNF152 antibodies alongside cytokine assays in experimental mouse models of inflammation to correlate RNF152 expression or function with cytokine production.

Research has demonstrated that RNF152-deficient mice produce significantly less IL-6 and TNFα in response to LPS challenge and are more resistant to LPS-induced lethal endotoxemia . When designing in vivo experiments, it's important to note that RNF152 deficiency specifically affects MyD88-dependent signaling pathways (like those downstream of IL-1R, TLR2, and TLR4) but not TRIF-dependent pathways (like TLR3-mediated signaling) . This specificity should be considered when selecting appropriate inflammatory models and stimuli.

What troubleshooting approaches can resolve contradictory results when using RNF152 antibodies?

When facing contradictory results with RNF152 antibodies, researchers should consider these methodological troubleshooting approaches:

• Antibody validation and specificity testing:

  • Use lysates from RNF152 knockout or knockdown cells as negative controls

  • Test multiple RNF152 antibodies targeting different epitopes (e.g., both F-4 and F-6 clones)

  • Perform peptide competition assays to confirm specificity

• Protocol optimization strategies:

  • Adjust antibody concentration and incubation conditions

  • Modify lysis buffers to ensure complete solubilization of membrane-associated RNF152

  • For western blotting, test different blocking agents to reduce background

• Context-dependent expression analysis:

  • Consider that RNF152 mRNA levels are upregulated following LPS stimulation , which may affect protein detection timing

  • Account for cell type-specific differences in RNF152 expression and function

  • Assess post-translational modifications that might mask antibody epitopes

• Functional validation approaches:

  • Complement antibody-based detection with functional assays

  • Use siRNA knockdown with different targeting sequences, as the efficiency of knockdown correlates with the degree of inhibition in downstream signaling

  • Compare results across multiple experimental systems (cell lines, primary cells, in vivo)

When analyzing contradictory results, it's important to consider that RNF152 has dual functions – both ubiquitination-dependent (in mTORC1 regulation) and ubiquitination-independent (in TLR/IL-1R signaling) , which might explain seemingly discrepant observations in different experimental contexts.

What are the optimal sample preparation methods for different applications of RNF152 antibodies?

For optimal results with RNF152 antibodies across different applications, researchers should consider these methodological approaches to sample preparation:

• For Western Blotting:

  • Lysis buffer: Use RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors when studying signaling pathways

  • Protein extraction: As RNF152 contains a transmembrane domain , consider membrane protein extraction protocols for complete solubilization

  • Sample denaturation: Standard denaturation at 95°C for 5 minutes in Laemmli buffer with β-mercaptoethanol

  • Protein loading: 20-50 μg total protein per lane for cell lysates

• For Immunoprecipitation:

  • Lysis buffer: Use milder NP-40 or Triton X-100 based buffers to preserve protein-protein interactions

  • Pre-clearing: Pre-clear lysates with appropriate control IgG to reduce non-specific binding

  • Antibody amount: Typically 2-5 μg of RNF152 antibody per 500 μg of total protein

  • Considerations for detecting interaction partners: When studying MyD88 interactions, crosslinking approaches may help capture transient interactions

• For Immunofluorescence:

  • Fixation: 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilization: 0.1% Triton X-100 for cytoplasmic proteins

  • Blocking: 5% BSA or normal serum in PBS

  • Antibody dilution: Typically 1:100 to 1:500 for primary RNF152 antibodies

• For ELISA:

  • Sample preparation: Cell lysates prepared in PBS with protease inhibitors

  • Concentration: Determine optimal coating concentration through titration

  • Blocking: 3-5% BSA or milk in PBS-T

  • Detection: HRP-conjugated RNF152 antibodies provide direct detection options

The optimization of these protocols is essential as RNF152 exhibits both membrane localization and involvement in cytoplasmic signaling complexes , which may require different extraction and preservation approaches depending on the specific aspect of RNF152 biology being investigated.

How can researchers effectively validate RNF152 antibody specificity for experimental applications?

To ensure the reliability of research findings, proper validation of RNF152 antibody specificity is crucial. Researchers should implement these methodological validation approaches:

• Genetic validation approaches:

  • Use lysates from RNF152 knockout mice or CRISPR/Cas9 engineered knockout cell lines as negative controls

  • Employ siRNA knockdown with varying efficiencies to demonstrate correlation between knockdown level and signal reduction

  • Use overexpression systems with tagged RNF152 to confirm co-localization with antibody signal

• Biochemical validation methods:

  • Perform peptide competition assays using the immunizing peptide

  • Test antibody reactivity against recombinant RNF152 protein

  • Compare results from antibodies targeting different epitopes of RNF152 (e.g., F-4 and F-6 clones)

• Cross-species reactivity assessment:

  • Validate antibody performance across mouse, rat, and human samples as claimed by manufacturers

  • Account for potential species-specific differences in RNF152 sequence and expression

• Application-specific validation:

  • For Western blotting: Confirm the detection of a single band at the expected molecular weight (~23 kDa for human RNF152)

  • For immunoprecipitation: Verify enrichment of RNF152 in IP fractions compared to input

  • For immunofluorescence: Demonstrate co-localization with known membrane markers or subcellular compartments where RNF152 is expected

When interpreting validation results, researchers should be aware that RNF152 expression may be dynamically regulated in response to stimuli such as LPS , which could affect detection levels under different experimental conditions.

What are the recommended approaches for studying RNF152 interactions with mTOR signaling components?

For investigating RNF152's interactions with mTOR signaling components, researchers should consider these methodological approaches:

• Co-immunoprecipitation strategies:

  • Use RNF152 antibodies to immunoprecipitate complexes, followed by immunoblotting for mTOR components

  • Perform reverse co-IP using antibodies against mTOR components and blotting for RNF152

  • Include detergent conditions that preserve membrane-associated complexes, as RNF152 contains a transmembrane domain

• Ubiquitination analysis of mTOR components:

  • Immunoprecipitate potential substrates (such as RagA) followed by ubiquitin immunoblotting

  • Use K63-linkage specific ubiquitin antibodies, as RNF152 has been shown to mediate K63-linked polyubiquitination of RagA

  • Perform in vitro ubiquitination assays with recombinant RNF152 and purified mTOR pathway components

• Functional analysis approaches:

  • Monitor mTORC1 activity (phosphorylation of S6K, 4E-BP1) in wild-type versus RNF152-deficient cells

  • Assess amino acid sensing pathways that regulate mTORC1 through the Rag GTPases

  • Combine RNF152 manipulation with mTOR inhibitors (rapamycin, Torin) to identify epistatic relationships

• Localization studies:

  • Use immunofluorescence to analyze co-localization of RNF152 with mTOR at the lysosomal surface

  • Employ subcellular fractionation followed by immunoblotting to detect compartment-specific interactions

  • Perform proximity ligation assays to visualize direct interactions in situ

Research indicates that RNF152 negatively regulates mTORC1 signaling through K63-linked polyubiquitination of RagA , suggesting an important role in nutrient sensing and cellular metabolism. When designing experiments, researchers should consider that RNF152 has dual roles in both mTOR regulation and inflammatory signaling, which might influence experimental outcomes depending on the cellular context and stimulation conditions.

What emerging applications of RNF152 antibodies show promise for advancing our understanding of inflammatory disorders?

Several emerging applications of RNF152 antibodies show significant potential for advancing our understanding of inflammatory disorders:

• Single-cell analysis approaches:

  • Integration of RNF152 antibodies into CyTOF (mass cytometry) panels to analyze expression patterns across immune cell subpopulations in inflammatory disease samples

  • Single-cell Western blotting to examine cell-to-cell variability in RNF152 expression and its correlation with inflammatory markers

  • Imaging mass cytometry to map RNF152 distribution in inflamed tissues with spatial resolution

• Therapeutic target validation strategies:

  • Use of RNF152 antibodies to validate it as a potential therapeutic target in inflammatory disease models

  • Correlation of RNF152 expression or activity with disease severity in clinical samples

  • Development of neutralizing antibodies against RNF152 as potential therapeutic tools

• Biomarker development applications:

  • Assessment of RNF152 levels in patient samples from inflammatory disorders to evaluate its potential as a biomarker

  • Correlation of RNF152 expression with treatment response to establish predictive biomarkers

• Systems biology integration:

  • Combination of RNF152 antibody-based proteomics with transcriptomics to create multi-omics profiles of inflammatory conditions

  • Network analysis to position RNF152 within the broader context of inflammatory signaling networks

Research has shown that RNF152-deficient mice are more resistant to LPS-induced lethal endotoxemia , suggesting that modulating RNF152 activity could potentially mitigate excessive inflammatory responses in sepsis and related disorders. The specificity of RNF152 for MyD88-dependent (but not TRIF-dependent) inflammatory signaling offers the possibility of selective intervention in specific inflammatory pathways, which could be particularly valuable for precision medicine approaches to inflammatory disorders.

How can RNF152 antibodies be utilized to explore the interplay between inflammation and metabolism?

To investigate the interplay between inflammation and metabolism involving RNF152, researchers should consider these methodological approaches:

• Dual pathway analysis strategies:

  • Use RNF152 antibodies in combination with metabolic and inflammatory signaling markers to simultaneously track both pathways

  • Apply multiplexed immunofluorescence to visualize co-localization of RNF152 with both mTOR components and TLR/IL-1R signaling molecules

  • Employ phospho-flow cytometry with RNF152 staining to correlate its expression with activation of both metabolic and inflammatory signaling pathways

• Nutrient response analysis:

  • Monitor RNF152 expression and localization changes during nutrient stress and inflammatory challenges

  • Investigate how amino acid availability affects RNF152's role in both mTORC1 regulation and inflammatory responses

  • Examine RNF152-dependent inflammatory responses under different metabolic conditions (glucose restriction, fatty acid oxidation, etc.)

• Metabolic phenotyping approaches:

  • Compare metabolic profiles of wild-type and RNF152-deficient immune cells during inflammatory activation

  • Assess glycolytic shift and mitochondrial function in relation to RNF152 expression during inflammation

  • Use metabolic flux analysis combined with RNF152 expression modulation to determine its impact on metabolic reprogramming during inflammation

• In vivo metabolic-inflammatory models:

  • Utilize RNF152 antibodies in models of metabolic inflammation (e.g., diet-induced obesity with inflammatory complications)

  • Analyze tissue-specific RNF152 expression in relation to both metabolic parameters and inflammatory markers

RNF152's dual role in mTORC1 regulation (through RagA ubiquitination) and MyD88-dependent inflammatory signaling positions it at a potential nexus between metabolism and inflammation. This intersection is particularly relevant for understanding conditions like metabolic syndrome, type 2 diabetes, and obesity-associated inflammation, where metabolic dysregulation and chronic inflammation are interconnected.

What methodological approaches are recommended for exploring RNF152's potential roles in cancer biology?

For investigating RNF152's potential roles in cancer biology, researchers should implement these methodological approaches:

• Expression analysis in cancer tissues:

  • Use RNF152 antibodies for immunohistochemical analysis of tumor microarrays spanning different cancer types

  • Correlate RNF152 expression with clinical parameters, including tumor stage, grade, and patient survival

  • Perform comparative analysis between tumor and adjacent normal tissues

• Functional studies in cancer models:

  • Manipulate RNF152 expression (knockdown/overexpression) in cancer cell lines and assess effects on:

    • Proliferation, apoptosis, and cell cycle progression

    • Migration and invasion capabilities

    • Resistance to chemotherapeutic agents

  • Use RNF152 antibodies to monitor protein expression in these models

• Pathway analysis in cancer context:

  • Investigate the impact of RNF152 on both mTOR signaling (a pathway frequently dysregulated in cancer) and inflammatory pathways within the tumor microenvironment

  • Examine RNF152's potential role in cancer-associated inflammation

  • Assess whether RNF152's E3 ligase activity targets tumor suppressors or oncogenes for ubiquitination

• In vivo cancer models:

  • Generate xenograft models with RNF152-manipulated cancer cells

  • Use RNF152 antibodies for immunohistochemical analysis of tumor tissues

  • Correlate RNF152 expression with tumor growth, metastasis, and response to therapy

The gene encoding RNF152 is located on human chromosome 18, a region associated with various cancers . Given RNF152's roles in both mTOR signaling and inflammatory responses, it may influence cancer development through multiple mechanisms: mTOR dysregulation is linked to cancer cell metabolism and growth, while inflammation can promote tumor development and progression. The ability to monitor RNF152 expression and function using specific antibodies provides valuable tools for exploring these potential connections in cancer biology.

How can RNF152 antibodies be incorporated into high-throughput screening approaches for drug discovery?

For incorporating RNF152 antibodies into high-throughput screening (HTS) for drug discovery, researchers should consider these methodological approaches:

• Cell-based screening platforms:

  • Develop reporter cell lines expressing RNF152-dependent readouts (e.g., NF-κB luciferase reporters)

  • Use automated immunofluorescence with RNF152 antibodies to screen for compounds that alter its expression, localization, or co-localization with binding partners

  • Implement high-content screening to simultaneously assess multiple parameters (RNF152 expression, signaling pathway activation, cell viability)

• E3 ligase activity screening:

  • Develop ELISA-based ubiquitination assays using RNF152 antibodies to capture the protein

  • Implement TR-FRET (time-resolved fluorescence energy transfer) assays combining RNF152 antibodies with ubiquitin detection

  • Screen for compounds that modulate RNF152's E3 ligase activity toward specific substrates

• Protein-protein interaction modulator screening:

  • Establish AlphaScreen or ELISA-based assays to detect RNF152 interactions with MyD88

  • Screen for compounds that enhance or disrupt these interactions

  • Validate hits using orthogonal assays such as co-IP with RNF152 antibodies

• Target engagement validation:

  • Develop cellular thermal shift assays (CETSA) using RNF152 antibodies to confirm direct binding of compounds to RNF152

  • Implement in-cell target engagement assays to validate compounds in a physiological context

Given that RNF152 positively regulates TLR/IL-1R-mediated inflammatory responses , compounds targeting RNF152 could have therapeutic potential for inflammatory disorders. Conversely, enhancers of RNF152 activity might boost antimicrobial defense mechanisms. The availability of highly specific RNF152 antibodies in various conjugated forms (FITC, PE, HRP) facilitates the development of diverse HTS platforms tailored to different drug discovery objectives.

What are the best practices for using RNF152 antibodies in combination with proteomics approaches?

For optimal integration of RNF152 antibodies with proteomics approaches, researchers should implement these methodological best practices:

• Immunoprecipitation-mass spectrometry (IP-MS) strategies:

  • Use RNF152 antibodies for immunoprecipitation followed by mass spectrometry to identify interacting proteins

  • Implement RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins) protocols for higher sensitivity

  • Compare interactomes under different conditions (e.g., before and after inflammatory stimulation)

  • Use label-free quantification or SILAC approaches for quantitative analysis of dynamic interactors

• Ubiquitinome analysis:

  • Combine RNF152 antibody-based purification with ubiquitin remnant profiling

  • Compare ubiquitination profiles between wild-type and RNF152-deficient cells to identify substrates

  • Focus on K63-linked ubiquitination sites, as RNF152 has been shown to mediate K63-linked polyubiquitination

• Proximity-dependent labeling:

  • Generate BioID or APEX2 fusion constructs with RNF152 for proximity labeling

  • Validate proximity labeling results using RNF152 antibodies in co-IP or co-localization studies

  • Focus analysis on protein networks related to both inflammatory signaling and mTOR regulation

• PTM mapping:

  • Use RNF152 antibodies to immunoprecipitate the protein for post-translational modification mapping

  • Investigate how PTMs on RNF152 might regulate its dual functions in inflammatory signaling and mTOR regulation

  • Develop phospho-specific or ubiquitin-specific RNF152 antibodies based on identified modification sites

When designing proteomics experiments, researchers should consider RNF152's membrane localization and ensure extraction methods compatible with membrane proteins. Additionally, the dynamic regulation of RNF152 expression in response to stimuli such as LPS suggests that temporal considerations are important when designing proteomics experiments to capture the full spectrum of RNF152 interactions across different cellular states.

What approaches are recommended for studying RNF152 in primary immune cells and tissue samples?

For investigating RNF152 in primary immune cells and tissue samples, researchers should consider these methodological approaches:

• Isolation and culture of primary immune cells:

  • Develop optimized protocols for RNF152 detection in various primary immune cell types (macrophages, dendritic cells, T cells, B cells)

  • Adjust fixation and permeabilization conditions for each cell type to optimize antibody accessibility

  • Consider cell type-specific expression levels when determining antibody concentrations

  • Use magnetic bead-based or flow cytometry-based cell sorting to isolate specific immune cell populations prior to RNF152 analysis

• Ex vivo stimulation and analysis:

  • Establish standardized stimulation protocols with TLR ligands (LPS, PGN) and cytokines (IL-1β)

  • Use RNF152 antibodies in combination with phospho-flow cytometry to simultaneously analyze signaling pathway activation

  • Implement multiplexed cytokine analysis to correlate RNF152 expression with functional outputs

  • Consider the timing of analysis, as RNF152 mRNA is upregulated following LPS stimulation

• Tissue immunohistochemistry optimization:

  • Optimize antigen retrieval methods for different tissue types

  • Implement multiplexed immunofluorescence to co-localize RNF152 with cell type-specific markers

  • Use tissue clearing techniques for thick section or whole-mount imaging with RNF152 antibodies

  • Compare staining patterns between inflamed and normal tissues

• Single-cell analysis in tissues:

  • Develop protocols for single-cell suspension preparation that preserve RNF152 epitopes

  • Optimize staining protocols for flow cytometry using commercially available conjugated RNF152 antibodies

  • Consider implementing imaging mass cytometry for spatial analysis of RNF152 in tissue context

Research has shown that RNF152 deficiency affects inflammatory responses in multiple primary cell types, including MEFs, BMDMs, and BMDCs . When studying RNF152 in these contexts, researchers should be aware of potential differences in expression levels and functional roles across different immune cell populations. Additionally, the differential effects of RNF152 deficiency on MyD88-dependent versus TRIF-dependent pathways suggest that stimulus selection is critical when designing experiments to probe RNF152 function in primary cells.