rnf213a Antibody

What is RNF213 and what are its key structural domains?

RNF213 is a large E3 ubiquitin ligase with a molecular weight of 591 kDa, located in the cytosol and perinuclear space. Structurally, it contains a C3HC4-type RING finger domain with E3 ubiquitin ligase activity and six AAA+ adenosine triphosphatase (ATPase) domains. It is notable for being the only known protein with both AAA+ ATPase and ubiquitin ligase activities . This unique combination of enzymatic activities suggests complex regulatory functions that may be uniquely controlled through antibody-based research approaches.

How does RNF213 function in immune regulation?

RNF213 plays a critical role in immune regulation by specifically promoting regulatory T (Treg) cell differentiation in CD4+ T cells. Mechanistically, RNF213 interacts with Forkhead Box Protein O1 (FOXO1) and promotes nuclear translocation of FOXO1 by facilitating K63-linked ubiquitination . This process is crucial for proper Treg differentiation and function. Research demonstrates that RNF213 deficiency leads to decreased Foxp3+ CD4+ T cells and reduced IL-10 production under Treg skewing conditions, confirming its essential role in Treg development .

What is the significance of RNF213 in antimicrobial responses?

Recent studies have established RNF213 as an important antimicrobial protein. It plays significant roles in fighting against various microbial infections, including viruses, parasites, bacteria, and chlamydiae . In viral infection studies, RNF213-deficient mice showed increased susceptibility to Rift Valley Fever Virus (RVFV), while mice overexpressing RNF213 demonstrated enhanced resistance and reduced infection symptoms . Additionally, RNF213 expression is significantly upregulated in experimental animals upon injection with highly pathogenic strains of avian influenza .

What criteria should researchers consider when selecting an anti-RNF213 antibody?

When selecting an anti-RNF213 antibody, researchers should consider:

• Antibody Type: Options include rabbit monoclonal (e.g., Anti-RNF213 Antibody, clone 1D10 ZooMAb ) or mouse monoclonal (e.g., Anti-RNF213 Antibody, clone 1C9 )

• Epitope Specificity: Verify the antibody targets the region of interest (e.g., the 1D10 clone targets an epitope within 24 amino acids from the N-terminal half )

• Validated Applications: Confirm the antibody is validated for your intended application (Western blotting, immunocytochemistry, immunohistochemistry, ELISA)

• Species Reactivity: Ensure compatibility with your experimental model

• Performance Documentation: Review quality control testing data from the manufacturer

The selection should be driven by your specific experimental questions and techniques rather than convenience or cost factors.

How should researchers validate anti-RNF213 antibodies before experimental use?

A comprehensive validation protocol for anti-RNF213 antibodies should include:

• Positive and Negative Controls:

  • Positive: Validated cell lines known to express RNF213 (e.g., A549, HUVEC, A431 )

  • Negative: RNF213 knockout cells or RNF213 siRNA-treated cells

• Dilution Optimization:

  • Western Blotting: Start with recommended dilution (e.g., 1:1,000 )

  • Immunocytochemistry: Begin with 1:100 dilution

  • Test multiple dilutions to determine optimal signal-to-noise ratio

• Specificity Testing:

  • Peptide competition assay with RNF213 peptide antigen

  • Affinity binding assay (e.g., verify KD values in the range of 10^-8 as reported )

• Cross-validation:

  • Compare results from at least two different anti-RNF213 antibodies

  • Correlate protein detection with mRNA expression data

How does RNF213 deficiency impact experimental autoimmune encephalomyelitis (EAE) models?

RNF213 deficiency significantly exacerbates experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Research demonstrates that RNF213-deficient mice develop more severe EAE with higher clinical scores and increased proinflammatory cytokines (IFN-γ, IL-17A, GM-CSF) alongside decreased IL-10 in serum compared to wild-type controls .

Analysis of the central nervous system (CNS) in these models reveals several key findings:

These findings highlight RNF213's critical role in regulating autoimmune responses through T cell differentiation and function .

What is the mechanism by which RNF213 facilitates Treg cell differentiation?

RNF213 promotes Treg cell differentiation through a specific molecular mechanism involving FOXO1. The process involves:

• Direct Interaction: RNF213 physically interacts with FOXO1, a critical transcription factor for Treg development

• Post-translational Modification: RNF213 catalyzes K63-linked ubiquitination of FOXO1

• Nuclear Translocation: This ubiquitination promotes FOXO1 nuclear translocation

• Transcriptional Activation: Nuclear FOXO1 then activates Foxp3 expression

• Treg Differentiation: Increased Foxp3 expression drives Treg cell differentiation

This mechanism is further supported by experiments showing that reintroduction of RNF213 into RNF213-deficient CD4+ T cells restores Treg differentiation capacity , providing a direct causative link between RNF213 activity and Treg development.

How does IFN-β treatment influence RNF213 expression and function in multiple sclerosis models?

IFN-β treatment significantly impacts RNF213 expression and function in multiple sclerosis contexts. Research demonstrates that:

• Induction Relationship: RNF213 expression in CD4+ T cells is directly induced by IFN-β treatment

• Therapeutic Connection: RNF213 exerts a crucial role in the therapeutic efficacy of IFN-β for multiple sclerosis

• Mechanistic Pathway: IFN-β → RNF213 upregulation → Enhanced Treg differentiation → Reduced autoimmune inflammation

• Clinical Relevance: This pathway provides a molecular explanation for one mechanism by which IFN-β therapy benefits MS patients

This relationship suggests that monitoring RNF213 expression levels could potentially serve as a biomarker for predicting IFN-β treatment efficacy in MS patients, though this application requires further clinical validation.

What are the optimal protocols for using anti-RNF213 antibodies in Western blotting?

Based on validated protocols, the following methodology is recommended for optimal Western blotting with anti-RNF213 antibodies:

Sample Preparation:

• Extract proteins from relevant cells (A549, HUVEC, or A431 are validated positive controls )

• Determine protein concentration using a reliable assay (BCA or Bradford)

• Load 20-40 μg of total protein per lane

Western Blotting Protocol:

• Separate proteins using 6% SDS-PAGE (RNF213 is large at 591 kDa)

• Transfer to PVDF membrane at low current (30V) overnight at 4°C

• Block with 5% non-fat milk in TBST for 1 hour at room temperature

• Incubate with anti-RNF213 antibody at 1:1,000 dilution overnight at 4°C

• Wash 3x with TBST, 10 minutes each

• Incubate with appropriate secondary antibody (HRP-conjugated anti-rabbit or anti-mouse depending on primary)

• Develop using enhanced chemiluminescence with extended exposure time

Troubleshooting Tips:

• For poor signal: Decrease the dilution to 1:500 or use a more sensitive detection system

• For high background: Increase blocking time and add additional washing steps

• For non-specific bands: Validate with peptide competition assay or RNF213 knockout controls

How can researchers effectively use anti-RNF213 antibodies in immunocytochemistry studies?

For optimal immunocytochemistry using anti-RNF213 antibodies, follow this validated protocol:

Cell Preparation:

• Grow cells on sterile coverslips to 70-80% confluence

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes

Immunostaining Protocol:

• Block with 3% BSA in PBS for 1 hour at room temperature

• Incubate with anti-RNF213 antibody at 1:100 dilution overnight at 4°C

• Wash 3x with PBS, 5 minutes each

• Incubate with fluorophore-conjugated secondary antibody for 1 hour at room temperature

• Counterstain nuclei with DAPI

• Mount with anti-fade mounting medium

Visualization Controls:

• Include a no-primary antibody control

• Use RNF213 knockout or knockdown cells as negative controls

• Co-stain with markers for cytosolic and perinuclear regions to confirm expected localization

What experimental approach should be used to assess RNF213's E3 ubiquitin ligase activity?

To assess RNF213's E3 ubiquitin ligase activity, researchers should employ the following comprehensive approach:

In Vitro Ubiquitination Assay:

• Express and purify recombinant RNF213 or its RING domain

• Set up reaction with E1, E2 enzymes, ubiquitin, ATP, and target substrate (e.g., FOXO1)

• Incubate at 37°C for 1-2 hours

• Analyze by SDS-PAGE and immunoblot for ubiquitin and target protein

Cell-Based Ubiquitination Analysis:

• Co-transfect cells with HA-tagged ubiquitin, RNF213, and potential substrate

• Treat with proteasome inhibitor (MG132) for 4-6 hours

• Lyse cells under denaturing conditions

• Immunoprecipitate substrate of interest

• Immunoblot for HA to detect ubiquitination

K63-Linkage Specific Analysis for FOXO1:
Since RNF213 specifically promotes K63-linked ubiquitination of FOXO1 , use:

• K63-linkage specific ubiquitin antibodies

• K63R ubiquitin mutant as a negative control

• Mass spectrometry to identify ubiquitination sites

This multi-faceted approach will provide definitive evidence of RNF213's E3 ligase activity and specificity for K63-linked ubiquitination.

How can researchers address contradictory findings regarding RNF213's role in different immune contexts?

Contradictory findings regarding RNF213's immune roles can be methodically addressed through:

• Context-Specific Analysis:

  • Compare RNF213 function in different immune cell types simultaneously

  • Analyze timing of RNF213 activity during immune response phases

  • Assess tissue-specific effects using conditional knockout models

• Stimulation-Dependent Effects:

  • Systematically compare RNF213 function under different stimulation conditions

  • Test multiple concentrations and time courses of cytokine treatments

  • Examine combined effects of multiple stimuli

• Resolution Approach:

  • Design experiments with direct head-to-head comparisons

  • Standardize experimental conditions across laboratories

  • Account for genetic background variations in mouse models

  • Collaborate across research groups to validate findings

• Mechanism Dissection:

  • Differentiate E3 ligase versus ATPase functions using domain-specific mutants

  • Identify context-specific binding partners through unbiased proteomics

  • Map differential post-translational modifications under varying conditions

What are the emerging techniques for studying RNF213 that go beyond traditional antibody applications?

Several cutting-edge techniques are emerging for RNF213 research that extend beyond traditional antibody applications:

• CRISPR-Based Approaches:

  • CRISPR activation (CRISPRa) to enhance endogenous RNF213 expression

  • CRISPR interference (CRISPRi) for targeted repression

  • CRISPR-based tagging for live-cell visualization of endogenous RNF213

  • Domain-specific mutagenesis to dissect functional regions

• Proximity Labeling Technologies:

  • BioID or TurboID fusion with RNF213 to identify proximal interacting proteins

  • APEX2-based proximity labeling for temporal interaction dynamics

  • Split-BioID for identifying condition-specific interactions

• Advanced Imaging Techniques:

  • Super-resolution microscopy to visualize RNF213 subcellular localization

  • Live-cell imaging with fluorescent protein fusions to track dynamics

  • Förster resonance energy transfer (FRET) to detect direct protein interactions

• Single-Cell Technologies:

  • Single-cell RNA-seq to identify cell populations with differential RNF213 expression

  • Single-cell proteomics to correlate RNF213 with cellular phenotypes

  • Single-cell ubiquitinome analysis to identify cell-specific substrates

These emerging techniques offer powerful complementary approaches to traditional antibody-based methods, enabling more comprehensive understanding of RNF213 biology.