MIB2 Antibody

What is MIB2 and what is its primary function in cellular processes?

MIB2 functions as a RING-type E3 ubiquitin ligase that catalyzes the transfer of ubiquitin molecules to target proteins. Its primary functions include:

• Enhancement of NF-κB activation through auto-ubiquitination via Lys-63 linkage

• Catalyzing Lys-48-linked polyubiquitination of target proteins like CYLD, marking them for proteasomal degradation

• Regulation of inflammatory responses through modulation of the NF-κB signaling pathway

• Involvement in reproductive biology, specifically in oocyte meiosis and early developmental competence

MIB2 contains five conserved domains: two MIB/Herc domains, an ankyrin repeat domain, and two RING domains, each serving specific functions in protein-protein interactions and catalytic activity .

How does MIB2 interact with the NF-κB signaling pathway?

MIB2 enhances NF-κB signaling through multiple mechanisms:

• Direct interaction with and degradation of CYLD, a deubiquitinating enzyme that normally suppresses NF-κB activity

• Catalyzation of Lys-48-linked polyubiquitination of CYLD at residues Lys-338 and Lys-530, targeting it for proteasomal degradation

• Auto-ubiquitination through Lys-63 linkage, which appears to enhance its pro-inflammatory signaling capabilities

Knockout studies have demonstrated that Mib2-deficient mice exhibit reduced serum interleukin-6 (IL-6) levels and suppressed inflammatory responses in arthritis models, confirming its role as a positive regulator of inflammation .

What are the key domains of the MIB2 protein and what are their functions?

MIB2 contains five distinct functional domains with specific roles in its cellular activities:

Deletion studies have demonstrated that RING domain-deleted MIB2 mutants (MIB2ΔRING) relocalize to the nucleus instead of maintaining their normal cytoplasmic localization . The ankyrin repeat region specifically interacts with the third CAP domain (amino acids 287-589) of CYLD .

Western Blot Analysis

• Prepare cell lysates in Laemmli buffer and heat at 95°C for 5 minutes

• Separate proteins using 10% SDS-PAGE followed by transfer to PVDF membrane

• Block with 5% low-fat dry milk in PBST for 1 hour at room temperature

• Incubate with primary anti-MIB2 antibody (e.g., Abcam #A17829) overnight at 4°C

• Wash with PBST three times before incubation with HRP-conjugated secondary antibody

• Visualize using ECL Plus detection system, with tubulin as loading control

Immunoprecipitation

• Use rabbit polyclonal anti-MIB2 antibodies for pull-down experiments

• Both wild-type and catalytically inactive MIB2 proteins can be co-immunoprecipitated with AGIA-CYLD protein

• Endogenous MIB2-CYLD interactions can be detected using anti-MIB2 antibody immunoprecipitation followed by CYLD detection

Subcellular Localization

• Immunofluorescence studies show that MIB2 and CYLD co-localize in the cytoplasm

• RING domains are essential for cytoplasmic localization, as their deletion causes nuclear localization

How can researchers effectively validate the specificity of MIB2 antibodies?

A comprehensive validation approach for MIB2 antibodies should include:

• Knockdown validation: Perform siRNA-mediated knockdown using validated sequences (forward: 5′-GUCGCUGUGAUGUGAAUGUTT-3′, reverse: 5′-ACAUUCACAUCACAGCGACTT-3′) and confirm antibody signal reduction by Western blot

• Recombinant protein controls: Express tagged versions of MIB2 (e.g., Myc-tagged) for positive control samples

• Domain mapping experiments: Test antibody recognition against various MIB2 deletion mutants to confirm epitope specificity

• Cross-reactivity assessment: Test antibody against related E3 ligases, particularly MIB1, to ensure specificity

• Multiple application testing: Validate antibody performance across different techniques (Western blot, immunoprecipitation, immunofluorescence) to confirm consistent recognition patterns

Effects of MIB2 Knockdown:

• Increased CYLD protein levels due to reduced ubiquitin-mediated degradation

• Inhibition of NF-κB signaling leading to decreased inflammatory responses

• In oocytes: disruption of normal meiotic progression and impaired NSN-to-SN chromatin configuration transition

Effects of MIB2 Overexpression:

• Enhanced CYLD degradation, confirmed by decreased CYLD protein half-life in cycloheximide chase experiments

• Increased NF-κB activation and downstream inflammatory signaling

• Catalytically inactive MIB2 mutants fail to reduce CYLD levels or enhance NF-κB signaling, despite maintaining protein interaction capability

These effects highlight MIB2's dual role in inflammatory regulation and reproductive development processes.

How does MIB2 contribute to the ubiquitination and degradation of CYLD?

MIB2 mediates CYLD degradation through a specific ubiquitination mechanism:

• Initial interaction occurs between MIB2's ankyrin repeat domain and the third CAP domain (amino acids 287-589) of CYLD

• Following binding, MIB2 catalyzes the addition of Lys-48-linked polyubiquitin chains specifically at residues Lys-338 and Lys-530 of CYLD

• This specific Lys-48-linked ubiquitination (not Lys-63-linked) targets CYLD for proteasomal degradation

• Cycloheximide chase experiments confirm that MIB2 expression decreases CYLD's cellular half-life

• The CYLD-K338/530R mutant lacking the two ubiquitination sites remains stabilized even with MIB2 overexpression

This mechanism allows MIB2 to enhance NF-κB signaling by removing the inhibitory effect of CYLD, which normally suppresses NF-κB activity.

What techniques are most effective for studying MIB2-protein interactions?

Several complementary techniques have proven effective for investigating MIB2 interactions:

• AlphaScreen assay: A cell-free protein-protein interaction detection system that has successfully demonstrated MIB2-CYLD interactions in vitro, producing signal strength comparable to known interaction partners like NEMO-CYLD

• GST-pulldown experiments: Using GST-CYLD fusion proteins to capture MIB2, confirming direct interaction

• Co-immunoprecipitation: Both overexpressed and endogenous MIB2-CYLD interactions can be detected using anti-MIB2 antibodies followed by Western blot analysis

• Deletion mutant analysis: Creating domain-specific deletions of both MIB2 and interaction partners to map binding interfaces, as demonstrated with MIB2-CYLD interaction mapping

• Immunofluorescence co-localization: Visualization of MIB2 and binding partners in cellular compartments, confirming their cytoplasmic co-localization

How can MIB2 antibodies be used to investigate inflammatory pathways?

MIB2 antibodies can be applied in multiple ways to elucidate inflammatory mechanisms:

• NF-κB pathway activation assessment: Monitor MIB2-mediated degradation of CYLD and subsequent NF-κB activation using antibodies against both MIB2 and CYLD

• Inflammatory cytokine profiling: Correlate MIB2 expression levels with inflammatory cytokine production such as IL-6 in various disease models

• Ubiquitination analysis: Use specific antibodies against Lys-48- or Lys-63-linked polyubiquitin chains together with MIB2 immunoprecipitation to characterize ubiquitination patterns

• Therapeutic target validation: In models of inflammatory disease, MIB2 antibodies can assess expression levels before and after experimental treatments

• Patient sample analysis: Compare MIB2 levels in tissues from inflammatory disease patients versus healthy controls to establish clinical relevance

In arthritis models, Mib2-knockout mice showed reduced serum IL-6 and suppressed inflammatory responses, suggesting MIB2 as a potential therapeutic target .

What are the experimental considerations when studying MIB2 in oocyte biology?

When investigating MIB2 in reproductive contexts, researchers should consider:

• Animal model selection: Use appropriate mouse models such as ICR female mice (3-4 weeks old) housed under controlled conditions (12h/12h light/dark cycle, 22°C, 20-30% humidity)

• Microinjection protocols: For knockdown experiments, inject 2.5 pl MIB2 siRNA (1 mM) using a Narishige microinjector; for overexpression, inject 10 pl cRNA (10 ng/μl)

• Post-injection culture: Arrest oocytes at GV stage in M16 medium containing 2.5 μM milrinone for 10 hours before releasing from meiotic arrest

• Sample preparation for protein analysis: Pool sufficient oocytes (at least 100) and lyse in Laemmli buffer for Western blot analysis

• Phenotypic assessment: Evaluate chromatin configuration changes, spindle morphology, and developmental competence to comprehensively assess MIB2 function

These specialized techniques ensure meaningful results when studying MIB2's role in oocyte meiosis and developmental processes.

How do post-translational modifications affect MIB2 function and antibody recognition?

Post-translational modifications significantly impact both MIB2 activity and its detection:

• Auto-ubiquitination: MIB2 undergoes auto-ubiquitination through Lys-63-linked chains, which enhances its NF-κB activating capacity

• Epitope masking: Antibodies targeting regions containing ubiquitination sites may show reduced binding to highly modified MIB2

• Functional regulation: Modifications can affect MIB2's E3 ligase activity, protein interactions, and subcellular localization

When selecting antibodies, researchers should consider whether the epitope contains potential modification sites and implement appropriate controls to ensure consistent detection regardless of modification state.

How can researchers distinguish between MIB2 and related E3 ubiquitin ligases?

Differentiating MIB2 from related E3 ligases requires multiple approaches:

• Antibody specificity: Select antibodies that have been validated against both MIB1 and MIB2 to ensure no cross-reactivity

• Substrate specificity: MIB2 preferentially ubiquitinates CYLD at specific lysine residues (Lys-338 and Lys-530) , which can help distinguish its activity

• Domain-specific analysis: The ankyrin repeat region of MIB2 mediates specific protein interactions that may differ from related E3 ligases

• Genetic approaches: Design siRNA sequences targeting unique regions not conserved between related E3 ligases

• Functional assays: Compare phenotypes resulting from specific knockdown of different E3 ligases to identify non-redundant functions

Understanding these distinctions is crucial for accurate characterization of MIB2-specific biological functions versus those of related family members.