RNF220 Antibody

What is RNF220 and what are its primary cellular functions?

RNF220 is an E3 ubiquitin-protein ligase with multiple critical cellular functions. Its primary role involves mediating polyubiquitination of target proteins, thus regulating their stability and degradation. In neurons, RNF220 directly interacts with AMPA receptors (AMPARs) to facilitate their polyubiquitination, serving as a key regulator of excitatory synaptic activity . RNF220 specifically targets GluA1 and GluA2 receptor subunits but does not interact with NMDA receptor subunits or kainate receptors .

Beyond neuronal signaling, RNF220 maintains hindbrain Hox gene expression patterns , regulates dorsoventral patterning of the hindbrain , and influences oligodendroglial development . It also promotes ubiquitination and proteasomal degradation of SIN3B and regulates WDR5 protein levels through K48-linked polyubiquitination .

What types of RNF220 antibodies are currently available for research?

Several RNF220 antibodies have been developed for research applications:

• Rabbit Polyclonal RNF220 antibody (ab236992) - Suitable for immunohistochemistry on paraffin-embedded tissues (IHC-P) with human samples, targeting amino acids 350-500 of human RNF220

• Mouse Polyclonal RNF220 antibody (ab69357) - Appropriate for Western blotting (WB) and immunocytochemistry/immunofluorescence (ICC/IF) with human samples

It's important to note that commercial RNF220 antibodies have limitations for in vivo immunoprecipitation analysis. In several studies, researchers have used alternative approaches such as RNF220-BAP (biotin-accepting peptide) mouse models to examine endogenous interactions between RNF220 and its targets .

How should RNF220 immunohistochemistry protocols be optimized?

For optimal RNF220 immunohistochemistry results, the following protocol has been validated for the rabbit polyclonal antibody (ab236992):

• Dewax and hydrate paraffin-embedded tissue sections

• Perform antigen retrieval using high pressure in citrate buffer (pH 6.0)

• Block sections with 10% normal goat serum for 30 minutes at room temperature

• Incubate with primary antibody diluted in 1% BSA (1:500 dilution recommended) at 4°C overnight

• Detect with biotinylated secondary antibody

• Visualize using an HRP-conjugated SP system

For immunofluorescence in neural tissues, RNF220 has been successfully detected in both the cytoplasm of PDGFRα+ oligodendrocyte precursor cells and CC1+ oligodendrocytes, although with lower intensity in the nucleus .

How can RNF220 antibodies be utilized to investigate AMPAR ubiquitination?

To investigate RNF220-mediated AMPAR ubiquitination, researchers can employ several complementary approaches:

• Co-immunoprecipitation assays: Transfect cells with tagged RNF220 and AMPAR subunits, then perform reciprocal co-IPs to confirm direct interaction. Studies have demonstrated that RNF220 co-immunoprecipitates with GluA1 and GluA2 but not with GluK2 (a kainate receptor subunit) or NMDAR subunits .

• Ubiquitination assays: After co-transfecting RNF220 with AMPAR subunits, immunoprecipitate the receptor and probe with anti-ubiquitin antibodies. Compare ubiquitination levels between wild-type RNF220 and ligase-dead mutants (ΔRING or W539R) .

• Comparison in knockout models: Immunoprecipitate GluA1 or GluA2 from brain homogenates of RNF220 conditional knockout mice and control littermates to assess endogenous ubiquitination levels. Both GluA1 and GluA2 ubiquitination are markedly decreased in RNF220 cKO hippocampus and cerebral cortex, while their protein levels are increased .

• Mutational analysis: Identify specific ubiquitination sites by mutating lysine residues in AMPAR C-termini. Research has identified K823/K868 in GluA1 and K825/K848/K864 in GluA2 as critical ubiquitination sites .

What controls should be included when examining RNF220 function in knockout models?

When investigating RNF220 function using knockout models, proper experimental controls are essential:

• Genetic controls: Always use littermate controls for genetic studies to minimize background effects .

• Validation of knockout efficiency:

  • Confirm mRNA reduction via qPCR (RNF220 cKO shows 75-85% reduction in mRNA levels)

  • Verify protein reduction via Western blotting (RNF220 cKO shows 82-91% reduction in protein levels)

  • Validate using immunostaining in relevant tissues

• Rescue experiments: Demonstrate specificity by rescuing phenotypes through reintroduction of wild-type RNF220 .

• Functional controls: Include ligase-dead mutants (ΔRING or W539R) to distinguish between ubiquitin ligase-dependent and independent functions .

• For CRISPR-mediated knockouts: Validate guide RNA efficiency using single-strand annealing recombination-based luciferase assays and confirm protein reduction via immunoblotting .

How do disease-associated RNF220 mutations affect its ubiquitin ligase function?

Two neuropathology-associated RNF220 mutations (R363Q and R365Q) have been characterized with significant functional consequences:

These mutations significantly impair the ability of RNF220 to bind and ubiquitinate AMPARs, preventing the regulation of receptor surface expression and synaptic activity . Unlike wild-type RNF220, these mutants cannot reverse the enhanced surface expression of GluA1 receptors caused by CRISPR-mediated deletion of RNF220 in neurons .

What methods are available for detecting RNF220-mediated protein polyubiquitination?

Several complementary approaches can be used to detect and characterize RNF220-mediated protein polyubiquitination:

• Immunoprecipitation and ubiquitin detection:

  • Immunoprecipitate the target protein (e.g., GluA1, GluA2, WDR5)

  • Detect polyubiquitination using anti-ubiquitin antibodies (such as P4D1)

  • Compare ubiquitination levels between wild-type and RNF220 knockout/knockdown samples

• Ubiquitin linkage characterization:

  • Use linkage-specific ubiquitin mutants (e.g., K48R) to determine ubiquitination type

  • For WDR5, RNF220 specifically mediates K48-linked polyubiquitination, which typically leads to proteasomal degradation

• Proteasome inhibition:

  • Treat cells with MG132 to block proteasomal degradation

  • If RNF220-mediated ubiquitination targets proteins for degradation, MG132 will prevent the reduction in protein levels

• Mutational analysis of ubiquitination sites:

  • Systematically mutate conserved lysine residues to arginines

  • Test each mutant's susceptibility to RNF220-mediated ubiquitination

  • For WDR5, lysines K109, K112, and K120 were identified as critical ubiquitination sites

How can RNF220 antibodies be used to investigate hindbrain development?

RNF220 plays critical roles in hindbrain development that can be investigated using the following approaches:

• Expression pattern analysis:

  • Use immunofluorescence to map RNF220 expression in developing hindbrain

  • RNF220 shows restricted expression in the ventral half of the ventricular zone at E10.5

  • As development progresses, RNF220-expressing cells appear in the mantle zone outside the ventricular zone by E12.5

• Domain-specific effects in knockout models:

  • Compare progenitor domain markers between control and RNF220 knockout embryos

  • In RNF220 Nestin CKO embryos, significant alterations are observed in ventral progenitor domains:

    • Reduction of p1 and p2 domains (marked by Dbx1, Olig2, and Nkx6.1)

    • Ventral expansion of the Dbx1+ p0 domain

    • Dorsal expansion of the Nkx2.2+ p3 domain

    • Dorsal shift and expansion of the Olig2+ pMN domain

• Analysis of post-mitotic derivatives:

  • Examine cells derived from affected progenitor domains, including oligodendrocyte precursor cells and serotonergic neurons

• WDR5 regulation and Hox gene expression:

  • Investigate RNF220's interaction with WDR5 and subsequent effects on Hox gene regulation

  • RNF220 regulates WDR5 through K48-linked polyubiquitination

What are the key limitations of current RNF220 antibodies?

Current RNF220 antibodies have several important limitations researchers should consider:

• Immunoprecipitation limitations: Commercial RNF220 antibodies have limited effectiveness for in vivo immunoprecipitation analysis . Researchers have developed alternative approaches such as:

  • RNF220-BAP mouse models, where RNF220 is fused with a biotin-accepting peptide

  • Tagged RNF220 constructs for exogenous expression systems

• Species reactivity: The documented antibodies primarily react with human samples, which may limit their utility in animal models .

• Application restrictions: Individual antibodies are validated for specific applications:

  • ab236992 is suitable for IHC-P but not verified for WB or IF

  • ab69357 is validated for WB and ICC/IF but not for IHC-P

• Recognition regions: The immunogens used for antibody generation target specific regions (e.g., aa 350-500 for ab236992), which may affect detection of truncated or mutant forms of RNF220 .

How should surface expression of RNF220-regulated proteins be analyzed?

To analyze surface expression of RNF220-regulated proteins such as AMPARs, researchers can employ these methodologies:

• BS3 cross-linking-based immunoprecipitation analysis:

  • This technique differentiates between surface and intracellular protein pools

  • Studies show increased surface expression of both GluA1 and GluA2 in the hippocampus and cerebral cortex of RNF220 cKO mice

• Membrane-impermeable immunostaining:

  • For cultured neurons, use live immunostaining with antibodies against extracellular domains

  • This approach revealed that RNF220 depletion increases endogenous GluA1 levels on neuronal surfaces, which can be rescued by cotransfection of gRNA-resistant RNF220

• Quantitative comparison:

  • Compare relative surface expression levels between experimental conditions

  • For example, RNF220 gRNA increased surface GluA1 to 133.98% of control levels, while rescue with RNF220 reduced it to 79.70%

  • Overexpression of wild-type RNF220 reduces surface GluA1 to 65.80% of control, while ubiquitination-deficient mutants (W539R or ΔRING) show reduced suppression ability

How should RNF220 antibodies be validated for research applications?

Thorough validation of RNF220 antibodies ensures reliable experimental results:

• Specificity validation:

  • Test antibody in RNF220 knockout/knockdown models

  • Verify absence or reduction of signal in tissues from RNF220 conditional knockout mice

  • Confirm specificity using overexpression systems with tagged RNF220 constructs

• Cross-reactivity assessment:

  • Test in multiple species if cross-species applications are planned

  • Verify performance in relevant cell types and tissues

• Application-specific validation:

  • For Western blotting: Confirm detection of a band at the expected molecular weight

  • For immunohistochemistry: Compare with in situ hybridization patterns

  • For immunofluorescence: Validate subcellular localization patterns (RNF220 is mainly observed in the cytoplasm with lower intensity in the nucleus)

• Blocking peptide controls:

  • Use immunizing peptides to confirm signal specificity

  • Pre-absorb antibody with excess antigenic peptide to demonstrate specific binding

• Recombinant protein detection:

  • Test sensitivity and specificity using recombinant RNF220 protein at various concentrations

How is RNF220 involved in oligodendroglial development?

Recent research indicates that RNF220 plays important roles in oligodendroglial development:

• Expression pattern:

  • RNF220 mRNA and protein expression remain relatively constant in oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes (OLs)

  • RNF220 is mainly localized in the cytoplasm of PDGFRα+ OPCs and CC1+ OLs, with lower intensity in the nucleus

• Functional studies:

  • RNF220 is required for oligodendroglial development

  • Specific depletion in OL lineage cells affects development, though the detailed mechanisms require further investigation

• Relationship to neural tube patterning:

  • RNF220's role in ventral neural tube patterning may influence the development of oligodendrocyte precursor cells

  • In RNF220 Nestin CKO embryos, changes in progenitor domains affect oligodendrocyte precursor cells

What are the implications of RNF220's differential ubiquitination activities?

RNF220 exhibits diverse ubiquitination activities with distinct functional outcomes:

• Target-specific ubiquitination:

  • For AMPARs: RNF220 mediates polyubiquitination at specific lysine residues (K823/K868 in GluA1; K825/K848/K864 in GluA2)

  • For WDR5: RNF220 catalyzes K48-linked polyubiquitination at K109, K112, and K120

• Linkage specificity:

  • K48-linked polyubiquitination (as with WDR5) typically leads to proteasomal degradation

  • The search results don't specify the linkage type for AMPAR ubiquitination, though it affects receptor stability

• Domain requirements:

  • The RING domain is essential for RNF220's ubiquitin ligase activity

  • RING domain deletion (ΔRING) or point mutations (W539R) abolish ubiquitination activity

  • Disease-associated mutations (R363Q, R365Q) significantly reduce binding to AMPARs and subsequent ubiquitination

• Non-degradative roles:

  • Independent of its E3 ligase activity, RNF220 can act as a CTNNB1 stabilizer through USP7-mediated deubiquitination, promoting Wnt signaling

This differential activity suggests RNF220 operates through multiple molecular mechanisms depending on cellular context and binding partners.

How might RNF220 mutations relate to neurological disorders?

The identification of neuropathology-associated RNF220 mutations suggests important connections to neurological disorders:

• Functional consequences of known mutations:

  • R363Q and R365Q mutations significantly impair RNF220's ability to:

    • Bind to AMPA receptors (reduced to 11-22% of wild-type)

    • Mediate AMPAR ubiquitination (reduced to 30-34% of wild-type)

    • Regulate surface expression of GluA1 receptors

    • Modulate AMPAR-mediated excitatory postsynaptic currents

• Potential pathological mechanisms:

  • Altered AMPAR surface expression could disrupt excitatory synaptic transmission

  • Imbalanced excitatory/inhibitory signaling might contribute to seizures or cognitive dysfunction

  • Abnormal hindbrain development due to RNF220 dysfunction could underlie developmental disorders

• Research approaches:

  • Generate knock-in mouse models carrying disease-associated mutations

  • Perform electrophysiological and behavioral analyses of these models

  • Investigate whether RNF220 mutations are enriched in specific neurological disorders

What are the potential connections between RNF220's roles in development and neuronal signaling?

RNF220 functions in both neurodevelopmental processes and mature neuronal signaling, suggesting integrated roles:

• Developmental timing:

  • RNF220 shows restricted expression in the ventral half of the ventricular zone during embryonic development

  • As development progresses, expression expands to post-mitotic neurons

• Integration of functions:

  • RNF220's regulation of Hox genes and neural tube patterning establishes the foundation for proper circuit formation

  • Its later role in AMPAR ubiquitination fine-tunes synaptic transmission in those circuits

• Future research directions:

  • Investigate whether RNF220's developmental roles affect its later functions in synaptic transmission

  • Develop temporally controlled conditional knockout models to separate early developmental effects from adult functions

  • Examine whether developmental abnormalities in RNF220 mutants predispose to synaptic dysfunction

The dual role of RNF220 in development and neuronal signaling highlights the importance of studying this protein across multiple time points and contexts to fully understand its contribution to brain function and dysfunction.