RNF144B Antibody

What is RNF144B and why is it important in immunological research?

RNF144B (Ring Finger Protein 144B) is an E3 ubiquitin ligase that plays critical roles in regulating innate immune responses. It has several aliases including p53RFP, IBRDC2, PIR2, and bA528A10.3 . RNF144B has emerged as a significant research target because it functions as a negative regulator of inflammation through multiple mechanisms:

• Inhibits LPS-induced inflammatory responses by interacting with TBK1 (TANK binding kinase 1)

• Negatively regulates antiviral immunity by targeting MDA5 (melanoma differentiation-associated protein 5) for autophagic degradation

• Suppresses inflammatory responses during sepsis, potentially protecting against cardiac dysfunction

The protein contains RING finger domains and an IBR (in between RING) domain, which are critical for its E3 ligase activity and protein interactions. Understanding RNF144B function has important implications for research into inflammatory diseases, antiviral responses, and sepsis.

What are the most common applications for RNF144B antibodies in research?

RNF144B antibodies are utilized across multiple research applications to study protein expression, localization, and interactions:

These applications have been instrumental in elucidating RNF144B's role in immune signaling pathways and inflammatory responses.

What are the key considerations when selecting an RNF144B antibody for my experiment?

When selecting an RNF144B antibody, researchers should consider several factors that can significantly impact experimental outcomes:

• Target epitope location: Different antibodies target distinct regions of RNF144B:

  • N-terminal region (AA 1-100, AA 1-101)

  • Central region (AA 90-118)

  • C-terminal region (AA 215-264, AA 218-303)

  • The epitope location may affect detection of specific protein interactions or post-translational modifications

• Species reactivity: Available antibodies show variable cross-reactivity:

  • Human-specific antibodies

  • Human and mouse cross-reactive antibodies

  • Broader reactivity across species (human, mouse, cow, dog, etc.)

• Clonality and validation:

  • Most commercial RNF144B antibodies are rabbit polyclonal

  • Validation methods vary by vendor (Western blot, IHC tissue arrays, recombinant protein arrays)

  • Some antibodies have been validated in published research

• Purification method:

  • Protein A column purification followed by peptide affinity purification

  • Antigen affinity chromatography purification

Select antibodies based on your specific experimental requirements, including application, species of interest, and specific protein domains you wish to study.

How can RNF144B antibodies be optimized for detecting protein-protein interactions in innate immune signaling?

Detecting RNF144B's interactions with its binding partners requires specific optimization strategies:

For TBK1-RNF144B interaction studies:

• Buffer optimization: Use buffers containing 0.1M Tris, 0.1M Glycine with 10% Glycerol (pH 7.0-7.4) to preserve protein interactions .

• Co-IP protocol refinement:

  • RNF144B has been shown to interact with the scaffold/dimerization domain (SDD) of TBK1 through its IBR domain

  • Crosslinking with DSP (dithiobis(succinimidyl propionate)) at 1-2 mM for 30 minutes can stabilize transient interactions

  • Include phosphatase inhibitors to preserve phosphorylation states of TBK1

• Sequential immunoprecipitation approach:

  • First IP with anti-TBK1 antibody

  • Elute under mild conditions

  • Second IP with anti-RNF144B antibody (26306-1-AP or equivalent)

  • This two-step approach increases specificity of detection

For MDA5-RNF144B studies:

• Autophagic degradation analysis:

  • Include autophagy inhibitors (3-MA or bafilomycin A1) to block RNF144B-mediated MDA5

  • Use RNF144B antibodies that target regions outside the MDA5-binding domain to avoid epitope masking

• Domain-specific interaction mapping:

  • RNF144B targets the CARDs of MDA5

  • Use truncation mutants in conjunction with co-IP to map specific interaction domains

Importantly, viral infection or LPS stimulation significantly alters these interactions, so time-course experiments are essential for capturing the dynamic nature of these regulatory mechanisms.

What methodological approaches can resolve contradictory findings regarding RNF144B's E3 ligase activity in different immune contexts?

The literature contains apparently contradictory findings regarding RNF144B's E3 ligase dependency in different contexts. These can be addressed with these methodological approaches:

• Domain-specific mutagenesis analysis:

  • Generate RING domain mutants (C20A/C23A) to abolish E3 ligase activity

  • Compare with IBR domain mutants that maintain E3 activity but alter protein interactions

  • This distinguishes E3-dependent from interaction-dependent effects

• Context-specific signaling pathway analysis:

  • LPS-TBK1 regulation by RNF144B appears independent of E3 ligase activity

  • Antiviral regulation via MDA5 is dependent on E3 ligase activity

  • Use pathway-specific inhibitors in parallel experiments:

    Pathway	Inhibitor	Concentration Range
    TLR4/LPS	TAK-242	0.1-1 μM
    MDA5/RIG-I	BX795 (TBK1 inhibitor)	0.5-2 μM
    Autophagy	3-MA or Bafilomycin A1	5-10 mM or 50-100 nM

• Ubiquitination profiling:

  • Use ubiquitin linkage-specific antibodies (K48 vs. K63)

  • Compare ubiquitination patterns in different stimulation conditions:

    • LPS stimulation (0, 15, 30, 60, 120 min)

    • Viral infection (EMCV, VSV)

    • poly(I:C) stimulation (high vs. low molecular weight)

• In vitro reconstitution assays:

  • Purified components (E1, E2, RNF144B, substrate)

  • Allows direct assessment of E3 ligase activity toward different substrates

This systematic approach can reconcile seemingly contradictory findings by demonstrating that RNF144B employs different mechanisms depending on the specific immune context and signaling pathway.

How can researchers effectively use RNF144B antibodies when studying knockout or knockdown models?

When using RNF144B knockout/knockdown models, special considerations are necessary for antibody-based validation and experimental design:

• Knockout model validation strategies:

  • The existing RNF144B knockout mouse model deletes exons 2 and 3

  • Validation requires both genomic PCR and protein-level confirmation

  • Use antibodies targeting multiple epitopes to confirm complete protein absence

  • Include positive controls from wild-type tissues (heart tissue shows good expression)

• siRNA knockdown verification protocols:

  • Transfect cells with RNF144B-specific siRNA

  • Validate knockdown efficiency at both mRNA level (qPCR) and protein level (Western blot)

  • Optimal antibody dilutions for knockdown verification: 1:500-1:1000 for Western blot

  • Include time-course analysis (24, 48, 72 hours post-transfection)

• Cell-type specific considerations:

  • RNF144B expression varies significantly between cell types

  • Baseline expression levels in different cell types:

    Cell Type	Relative Expression	Detection Notes
    THP-1 (human monocytic)	Moderate, inducible	LPS-responsive
    BMDMs	High, inducible	Strong LPS induction
    MEFs	Moderate	Detectable in wild-type
    HEK293T	Low	Often used for overexpression

• Phenotypic analysis protocol optimizations:

  • For viral infection studies, measure:

    • Viral replication (plaque assays)

    • IFN-β production (ELISA, qPCR)

    • IRF3 phosphorylation (phospho-specific antibodies)

  • For inflammatory models:

    • Cytokine profiling (TNFα, IL-6, IFN-β)

    • TBK1 and IκBα phosphorylation status

    • NF-κB nuclear translocation (nuclear fractionation or IF)

These approaches enable rigorous validation of knockout/knockdown models and accurate interpretation of resulting phenotypes.

How can RNF144B antibodies be used to investigate its role in the autophagic degradation of MDA5?

Recent research has identified RNF144B as mediating autophagic degradation of MDA5, representing an important area for investigation using specialized antibody techniques:

• Sequential immunofluorescence protocol:

  • Double immunostaining for RNF144B and MDA5

  • Additional staining for autophagy markers (LC3, p62/SQSTM1)

  • Time-course analysis following viral infection or poly(I:C) stimulation

  • Confocal microscopy with colocalization analysis

• Proximity ligation assay (PLA) optimization:

  • Allows in situ detection of protein-protein interactions

  • Use anti-RNF144B (rabbit) and anti-MDA5 (mouse) primary antibodies

  • Secondary antibodies conjugated with PLA probes

  • Signal represents close proximity (<40 nm) between proteins

  • Include appropriate controls (single primary antibodies)

• Autophagy flux assessment:

  • Monitor MDA5 degradation in presence/absence of RNF144B

  • Use autophagy inhibitors (bafilomycin A1, chloroquine)

  • Western blot analysis with anti-MDA5 and anti-LC3 antibodies

  • Compare results between wild-type and RNF144B-deficient cells

• Domain-specific interaction mapping:

  • RNF144B specifically targets the CARDs of MDA5

  • Use deletion mutants of MDA5 (ΔCARD) to confirm interaction specificity

  • Immunoprecipitate with anti-RNF144B antibody and blot for MDA5 variants

This methodological approach provides a comprehensive analysis of how RNF144B regulates MDA5 through autophagic mechanisms, potentially revealing new therapeutic targets for modulating antiviral responses.

What are the technical challenges in detecting endogenous vs. overexpressed RNF144B and how can they be addressed?

Researchers face distinct challenges when detecting endogenous versus overexpressed RNF144B, requiring specific technical adaptations:

• Endogenous RNF144B detection optimization:

  • Baseline expression is often low in unstimulated cells

  • Enhance detection by:

    • Using signal amplification methods (HRP-conjugated polymers)

    • Longer primary antibody incubation (overnight at 4°C)

    • Concentrating protein samples (immunoprecipitation before Western blot)

    • Stimulating cells with LPS (2-6 hours) to upregulate RNF144B expression

• Overexpression artifacts and controls:

  • Tag interference issues:

    • N-terminal tags may interfere with RING domain function

    • C-terminal tags may affect protein localization

  • Include untagged controls and multiple tag positions

  • Verify function with complementation assays in knockout cells

  • Compare results between different expression levels

• Standardized comparison protocol:

  • Load endogenous and overexpressed samples in the same gel

  • Include concentration gradient of recombinant protein as standard curve

  • Apply quantitative Western blot techniques with fluorescent secondary antibodies

  • Optimize antibody dilutions separately for each condition:

    Condition	Recommended Dilution	Exposure Time
    Endogenous	1:500	Extended (3-5 min)
    Overexpressed	1:1000-1:3000	Brief (30 sec-1 min)

• Specificity validation in knockout systems:

  • Include RNF144B knockout/knockdown controls

  • Test multiple antibodies targeting different epitopes

  • Perform peptide competition assays to confirm specificity

  • Consider cross-reactivity with related family members (e.g., RNF144A)

These approaches allow researchers to accurately detect and compare RNF144B under different experimental conditions while avoiding common technical pitfalls that can lead to misinterpretation of results.

How should researchers design experiments to investigate the dual roles of RNF144B in both antiviral responses and inflammatory pathways?

RNF144B exhibits complex regulatory functions in both antiviral immunity and inflammatory pathways, requiring carefully designed experimental approaches to dissect these distinct but interconnected roles:

• Pathway-specific stimulation protocol:

  • Selective pathway activation:

    Pathway	Stimulus	Concentration	Duration
    MDA5 pathway	High MW poly(I:C) transfection	1-2 μg/ml	6-24h
    RIG-I pathway	Low MW poly(I:C) or 5'ppp-RNA	0.5-1 μg/ml	6-24h
    TLR4 pathway	LPS	100-500 ng/ml	2-24h
    TLR3 pathway	Poly(I:C) (extracellular)	10-25 μg/ml	6-24h

  • Measure pathway-specific outputs (IFN-β for antiviral, TNFα/IL-6 for inflammatory)

  • Compare responses in wild-type and RNF144B-deficient cells

• Time-resolved analysis of RNF144B interactions:

  • Investigate dynamic protein interactions over time:

    • Early phase (0-2h): Focus on TBK1-RNF144B interaction

    • Late phase (2-24h): Focus on MDA5-RNF144B interaction

  • Use sequential co-immunoprecipitation at defined timepoints

  • Analyze post-translational modifications of RNF144B throughout response

• Viral infection model selection:

  • RNA viruses that differentially engage antiviral pathways:

    Virus	Primary Sensor	RNF144B Effect
    EMCV	MDA5-dependent	Enhanced survival in KO mice
    VSV	RIG-I-dependent	Impaired replication in KO cells
    Influenza	RIG-I/TLR7	Not yet characterized

  • Compare with bacterial component stimulation (LPS, CpG)

  • Include readouts for both viral replication and inflammatory markers

• In vivo model validation strategy:

  • Use tissue-specific conditional knockout models:

    • Myeloid-specific (LysM-Cre) for inflammatory models

    • Systemic knockout for viral challenge models

  • Compare survival rates, viral loads, and cytokine profiles

  • Analyze tissue-specific effects (e.g., cardiac dysfunction in sepsis models)

  • Include both acute and chronic infection/inflammation models

This comprehensive experimental approach allows researchers to delineate the distinct mechanisms by which RNF144B regulates antiviral immunity versus inflammatory responses, potentially revealing new therapeutic opportunities for modulating these pathways independently.

What are the common technical issues when using RNF144B antibodies and how can they be resolved?

Researchers frequently encounter technical challenges when working with RNF144B antibodies. Here are systematic approaches to address these issues:

• High background in Western blots:

  • Potential causes and solutions:

    Cause	Solution
    Antibody concentration too high	Titrate antibody, starting at 1:1000 and adjust as needed
    Insufficient blocking	Extend blocking time to 2h; try 5% milk or 3-5% BSA in TBST
    Cross-reactivity	Pre-absorb antibody with cell lysate from RNF144B knockout cells
    Membrane overexposure	Reduce exposure time; use fluorescent secondaries for linear detection

• Poor signal-to-noise ratio in immunohistochemistry:

  • Optimization strategies:

    • Heat-mediated antigen retrieval with TE buffer pH 9.0 yields better results than citrate buffer

    • Extend primary antibody incubation to overnight at 4°C

    • Reduce antibody concentration (1:200-1:400) and increase incubation time

    • Use amplification systems (HRP-polymers) rather than increasing antibody concentration

    • Include appropriate positive tissue controls (human kidney tissue shows good expression)

• Failed co-immunoprecipitation experiments:

  • Troubleshooting approach:

    • Verify RNF144B expression in input samples

    • Use milder lysis conditions (avoid SDS, use NP-40 or Triton X-100)

    • Add protease and phosphatase inhibitors (especially for TBK1 interactions)

    • Consider crosslinking to stabilize transient interactions

    • Include stimulation conditions that enhance expression (LPS treatment for 4-6h)

• Inconsistent antibody performance between lots:

  • Quality control measures:

    • Always validate new lots against previous lots

    • Maintain positive control lysates from overexpression systems

    • Document optimal dilutions and conditions for each lot

    • Consider pooling antibodies from multiple lots for long-term projects

These systematic troubleshooting approaches help ensure consistent and reliable results when working with RNF144B antibodies across different experimental applications.

How can researchers validate the specificity of RNF144B antibodies in their experimental systems?

Rigorous validation of RNF144B antibody specificity is essential for generating reliable data. Implement these comprehensive validation strategies:

• Genetic validation approaches:

  • Use RNF144B knockout or knockdown models:

    • CRISPR-Cas9 generated knockout cell lines

    • siRNA/shRNA knockdown (verify >80% reduction)

    • Tissue from RNF144B knockout mice (heart tissue recommended)

  • Positive signal should be absent or significantly reduced

  • Include wild-type controls processed identically

• Molecular validation techniques:

  • Peptide competition assay:

    • Pre-incubate antibody with 5-10 fold excess of immunizing peptide

    • Compare with non-competed antibody

    • Specific signals should be eliminated or substantially reduced

  • Multiple antibody verification:

    • Test antibodies targeting different epitopes (N-terminal, central, C-terminal)

    • Compare detection patterns across applications

• Expression-based validation:

  • Overexpression systems:

    • Transfect with tagged RNF144B constructs (FLAG, HA, GFP)

    • Detect with both anti-tag and anti-RNF144B antibodies

    • Signals should co-localize in IF or show bands of appropriate size in WB

  • Stimulus-dependent expression:

    • Treat cells with LPS (100-500 ng/ml) for 4-6 hours

    • RNF144B expression should increase in response to stimulation

    • Verify by both qPCR and Western blot

• Cross-reactivity assessment:

  • Test on related proteins:

    • RNF144A (closest family member)

    • Other RING-IBR-RING (RBR) E3 ligases

  • Species cross-reactivity verification:

    • If using in multiple species, verify with species-specific positive controls

    • Sequence alignment of epitope regions across species can predict cross-reactivity

These validation approaches ensure that the observed signals truly represent RNF144B, providing a solid foundation for subsequent experimental investigations.

How might RNF144B antibodies contribute to understanding its potential therapeutic role in inflammatory and infectious diseases?

Recent discoveries about RNF144B's immunoregulatory functions suggest several promising research directions where specialized antibody applications could advance therapeutic development:

• Sepsis intervention research applications:

  • RNF144B deficiency exacerbates cardiac dysfunction and increases mortality in septic mice

  • Research opportunities:

    • Immunohistochemical mapping of RNF144B expression in patient tissues

    • Correlation studies between RNF144B levels and disease severity

    • Therapeutic testing using RNF144B-overexpressing models

    • Monitoring RNF144B as a potential biomarker for sepsis progression

• Antiviral immunity modulation:

  • RNF144B knockout mice show enhanced survival after EMCV infection

  • Antibody applications:

    • Screening for small molecules that modulate RNF144B-MDA5 interaction

    • Proximity ligation assays to monitor drug effects on protein interactions

    • Intracellular antibody delivery to disrupt specific domain interactions

    • Correlating RNF144B expression with viral susceptibility in patient cohorts

• Autoimmune disease connections:

  • RNF144B's role in limiting type I IFN suggests relevance to autoimmune disorders

  • Research directions:

    • Profiling RNF144B expression in autoimmune disease tissues

    • Investigating genetic variants using epitope-specific antibodies

    • Exploring RNF144B regulation of tonic IFN signaling in autoimmunity

    • Developing targeted delivery of RNF144B modulators to affected tissues

• Experimental therapeutic approaches:

  • Context-dependent modulation strategies:

    Disease Context	Therapeutic Approach	Antibody Application
    Viral infections	RNF144B inhibition	Monitor target engagement
    Sepsis/inflammation	RNF144B enhancement	Assess pathway modulation
    Autoimmunity	Context-specific regulation	Cell type-specific profiling

These emerging research directions highlight the dual role of RNF144B in different disease contexts and suggest that precise, context-dependent modulation of its activity could offer novel therapeutic strategies for both infectious and inflammatory diseases.

What new methodological approaches are being developed for studying RNF144B's E3 ligase activity in different physiological contexts?

Innovative methodological approaches are emerging to study RNF144B's E3 ligase activity and its context-dependent functions:

• Proximity-based enzymatic assays:

  • BioID or TurboID fusion proteins:

    • RNF144B fused to biotin ligase identifies proximal proteins

    • Allows mapping of substrates in different physiological contexts

    • Compare substrate profiles between viral infection and LPS stimulation

    • Validate hits with targeted co-IP and ubiquitination assays

  • Split-ubiquitin complementation:

    • Enables direct visualization of ubiquitination events in live cells

    • Monitor real-time dynamics of RNF144B-mediated ubiquitination

• Ubiquitin linkage-specific analysis:

  • Mass spectrometry approaches:

    • Ubiquitin-remnant profiling with K48/K63-specific antibodies

    • AQUA peptide standards for absolute quantification

    • Compare ubiquitination patterns in wild-type vs. RNF144B knockout cells

  • Linkage-specific reporters:

    • Fluorescent sensors for different ubiquitin chain types

    • Real-time monitoring of ubiquitination dynamics

• CRISPR-based functional screening:

  • Domain-focused mutagenesis screens:

    • Systematic mutation of RING and IBR domains

    • Correlation of E3 activity with functional outcomes

    • Identification of separation-of-function mutants (E3 activity vs. protein binding)

  • Pathway-specific reporter screens:

    • IFN-β or NF-κB luciferase reporters

    • Screen for residues critical for pathway regulation

• Structural biology approaches:

  • Cryo-EM analysis of RNF144B complexes:

    • RNF144B-TBK1 interactions

    • RNF144B-MDA5 complexes

    • Antibody fragment (Fab) co-crystallization to stabilize complexes

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

    • Map conformational changes upon substrate binding

    • Identify allosteric regulation mechanisms

These innovative methodological approaches will provide deeper insights into how RNF144B's E3 ligase activity is regulated in different physiological contexts and how it contributes to the differential regulation of antiviral versus inflammatory responses.

How can RNF144B antibodies be used to investigate its potential roles beyond innate immunity?

While RNF144B has been primarily studied in innate immunity, emerging evidence suggests broader functions that can be investigated using specialized antibody techniques:

• Cell death regulation exploration:

  • RNF144B has been implicated in regulating apoptosis through Bax

  • Research approaches:

    • Dual immunostaining for RNF144B and mitochondrial markers

    • Co-localization analysis during apoptotic stimulation

    • Tracking RNF144B translocation to mitochondria during cell death

    • Correlating RNF144B expression with apoptotic markers in disease tissues

• p53 pathway connections:

  • RNF144B (p53RFP) was originally identified as a p53-inducible gene

  • Investigation strategies:

    • ChIP assays to confirm p53 binding to RNF144B promoter

    • Expression correlation studies between p53 and RNF144B

    • Analysis of RNF144B in p53-dependent cellular responses

    • Potential roles in cancer through p53-mediated pathways

• Metabolic regulation possibilities:

  • E3 ligases often have roles in metabolic processes

  • Exploratory approaches:

    • Immunoprecipitation-mass spectrometry to identify novel interacting partners

    • Subcellular fractionation to determine localization in metabolic organelles

    • Metabolic profiling of RNF144B knockout vs. wild-type cells

    • Monitoring RNF144B expression in response to metabolic stress

• Tissue-specific expression analysis:

  • Comprehensive immunohistochemical profiling:

    Tissue Type	RNF144B Expression	Potential Function
    Immune cells	High, inducible	Inflammatory regulation
    Heart	Constitutive	Protection against dysfunction
    Kidney	Detectable	Not yet characterized
    Brain	Low/variable	Requires investigation

  • Correlation with tissue-specific phenotypes in knockout models

These research directions extend beyond RNF144B's established roles in immunity and could reveal unexpected functions in diverse physiological and pathological processes, potentially identifying new therapeutic opportunities.