znrf1 Antibody

What is ZNRF1 and what cellular functions does it perform?

ZNRF1 belongs to the largest class of RING-finger E3 ligases in mammals and was originally identified in injury-induced nerve cells. This protein contains both zinc finger and RING finger motifs and primarily localizes to endosome-lysosome compartments . ZNRF1 performs several critical cellular functions, including regulation of receptor trafficking, ubiquitination of target proteins, and modulation of immune responses. Specifically, it mediates TLR3 ubiquitination and trafficking to lysosomes for degradation, controls EGFR endocytic sorting, and influences neural cell differentiation . Recent research has also revealed that ZNRF1 plays a significant role in antiviral immunity, particularly against EMCV and SARS-CoV-2 infections .

What is the molecular weight of ZNRF1 protein and why might observed weights differ from calculated values?

The ZNRF1 protein has an observed molecular weight of approximately 24 kDa on Western blot analysis, although calculations based on amino acid sequence predict molecular weights of 23 kDa and 29 kDa . This discrepancy between observed and calculated molecular weights commonly occurs due to post-translational modifications like phosphorylation, glycosylation, or ubiquitination. In particular, ZNRF1's function as an E3 ubiquitin ligase may involve auto-ubiquitination, potentially affecting its migration pattern during gel electrophoresis. Additionally, different isoforms or proteolytically processed forms may contribute to varying molecular weights observed in experimental settings .

What types of ZNRF1 antibodies are available for research applications?

Commercially available ZNRF1 antibodies include polyclonal antibodies with reactivity to human, mouse, and rat samples . These antibodies are typically produced using recombinant fusion proteins of human ZNRF1 as immunogens. The antibodies are primarily recommended for Western blot applications at dilution ranges of 1:500-1:2000, though they may be optimized for additional applications depending on experimental needs . When selecting a ZNRF1 antibody, researchers should consider the specific epitope recognition, cross-reactivity with other species, and validated applications in published literature.

What are the optimal protocols for detecting ZNRF1 in different cell lines?

For optimal detection of ZNRF1 across various cell lines, a Western blot protocol using SDS-PAGE followed by immunoblotting with a validated ZNRF1 antibody is recommended. Based on published research, the following methodological considerations are important:

• Sample preparation: Extract proteins using lysis buffers containing protease inhibitors to prevent degradation.

• Protein loading: Load 20-40 μg of total protein per lane for adequate detection.

• Antibody dilution: Use ZNRF1 antibodies at 1:1000 dilution for optimal signal-to-noise ratio .

• Detection system: Use either chemiluminescence or fluorescence-based detection systems.

• Controls: Include positive controls (cell lines known to express ZNRF1) such as A549, HeLa, or RAW264.7 macrophages, as these have been validated in research studies .

ZNRF1 expression has been successfully detected in various cell types including macrophages (RAW264.7, BMDMs), epithelial cells (A549, H3255, HeLa, Calu-3), and fibroblasts (MEFs) .

How should researchers design experiments to study ZNRF1's interaction with binding partners?

When investigating ZNRF1's interactions with binding partners such as TLR3 or EGFR, researchers should employ:

• Co-immunoprecipitation (Co-IP): This technique effectively demonstrates protein-protein interactions. For ZNRF1-EGFR interactions, studies have shown that the zinc finger domain of ZNRF1 binds to the tyrosine kinase domain (TKD) of EGFR .

• Domain mapping: Create domain deletion mutants to identify specific interaction regions. For example, research has shown that deletion of the ZNRF1 zinc finger domain significantly impedes ZNRF1 binding to EGFR .

• Proximity ligation assays: These provide spatial resolution of protein interactions in situ.

• Functional validation: After identifying interactions, validate their significance through functional assays that demonstrate altered cellular responses when the interaction is disrupted .

What methods are effective for manipulating ZNRF1 expression in experimental systems?

Several approaches have proven effective for modulating ZNRF1 expression:

• CRISPR/Cas9 gene editing: This technique has been successfully used to generate ZNRF1 knockout cell lines, including human lung epithelial Calu-3 cells and A549 cells .

• RNA interference: shRNA against ZNRF1 has been effective in various cell lines including A549, H3255, and HeLa cells .

• Overexpression systems: Lentiviral vectors encoding Flag-tagged wild-type ZNRF1 or catalytically inactive mutants (such as ZNRF1 C184A) allow for reconstitution experiments in knockout backgrounds .

• Animal models: ZNRF1 knockout mice (Znrf1−/−) have been generated and characterized for in vivo studies of ZNRF1 function in immunity and disease models .

Each approach offers distinct advantages depending on the research question, with CRISPR providing complete protein elimination, while RNAi allows for temporal control and varying degrees of knockdown.

How does ZNRF1 regulate TLR3 trafficking and interferon responses?

ZNRF1 functions as a key negative regulator of TLR3-mediated immune responses through a sophisticated mechanism:

• Activation mechanism: Upon TLR3 activation, c-Src phosphorylates ZNRF1 at its 103rd tyrosine residue, which activates ZNRF1's E3 ligase activity .

• Receptor modification: Activated ZNRF1 associates with TLR3 and mediates K63-linked polyubiquitination at TLR3 K813 .

• Trafficking control: This ubiquitination promotes TLR3 trafficking to lysosomes for degradation, thereby limiting the duration of TLR3 signaling .

• Physiological consequences: ZNRF1 deficiency results in prolonged TLR3 signaling and increased type I interferon production. This has dual effects:

  • Enhanced resistance to viral infections like EMCV and SARS-CoV-2

  • Increased susceptibility to opportunistic bacterial infections due to excessive type I IFN responses and impaired lung tissue repair

To study this pathway, researchers should employ kinase inhibitors (for c-Src), ubiquitination assays, endosomal trafficking markers, and cytokine production measurements to comprehensively analyze ZNRF1's regulatory role.

What are the methodological approaches for studying ZNRF1's role in EGFR degradation?

To investigate ZNRF1's function in EGFR degradation, researchers should implement the following methodological approaches:

• Receptor degradation kinetics: Monitor EGFR protein levels following EGF stimulation in wild-type versus ZNRF1-deficient cells using pulse-chase experiments and Western blotting .

• Ubiquitination analysis: Perform in vivo ubiquitination assays to detect EGFR ubiquitination levels in the presence or absence of ZNRF1. This reveals that ZNRF1 mediates ubiquitination at distinct lysine residues compared to other E3 ligases like CBL .

• Endosomal trafficking visualization: Use immunofluorescence microscopy with markers for early endosomes (EEA1), late endosomes (Rab7), and lysosomes (LAMP1) to track EGFR movement through the endocytic pathway .

• Pharmacological interventions: Apply lysosomal inhibitors (Chloroquine) or proteasomal inhibitors (MG132) to distinguish between degradation pathways. Research has shown that ZNRF1-mediated EGFR degradation occurs primarily through the lysosomal pathway .

• Signaling output measurement: Assess downstream signaling pathways (MAPK, AKT) to determine functional consequences of altered EGFR degradation in ZNRF1-manipulated cells .

How can researchers distinguish between ZNRF1-mediated and CBL-mediated ubiquitination of EGFR?

Distinguishing between ZNRF1-mediated and CBL-mediated EGFR ubiquitination requires sophisticated experimental approaches:

• Site-specific ubiquitination analysis: Mass spectrometry-based approaches can identify specific lysine residues ubiquitinated by each E3 ligase. Research indicates that ZNRF1 and CBL mediate ubiquitination at distinct lysine residues on EGFR .

• Domain-specific mutants: Generate EGFR mutants lacking specific lysine residues to determine which are required for ZNRF1 versus CBL-mediated ubiquitination .

• Selective E3 ligase inhibition: Employ genetic knockouts or domain-specific mutants of either ZNRF1 or CBL to assess their individual contributions to EGFR ubiquitination patterns.

• Temporal analysis: Investigate the timing of ZNRF1 versus CBL recruitment to activated EGFR using time-course immunoprecipitation combined with Western blotting.

• Functional consequences: Examine differences in receptor trafficking, signaling duration, and cellular responses between ZNRF1-deficient, CBL-deficient, and double-deficient cells to understand their cooperative versus unique functions .

What are common challenges in ZNRF1 antibody validation and how can they be addressed?

Validating ZNRF1 antibodies presents several challenges that researchers should systematically address:

• Specificity verification: Confirm antibody specificity using multiple approaches:

  • Western blot analysis in ZNRF1 knockout or knockdown cells as negative controls

  • Testing across multiple cell lines with varying ZNRF1 expression levels

  • Peptide competition assays to verify epitope recognition

• Cross-reactivity assessment: Test antibody performance across species if working with model organisms, as commercially available antibodies show reactivity with human, mouse, and rat ZNRF1 .

• Batch-to-batch variation: When using polyclonal antibodies, significant variation can occur between production lots. Document lot numbers and validate each new batch against previous results.

• Detection of post-translational modifications: If studying phosphorylated ZNRF1 (e.g., Y103 phosphorylation by c-Src ), use phospho-specific antibodies and validate with phosphatase treatments.

• Optimization for specific applications: While ZNRF1 antibodies are validated for Western blotting (1:500-1:2000 dilution) , additional optimization may be required for immunoprecipitation, immunofluorescence, or flow cytometry applications.

What factors may lead to inconsistent results when studying ZNRF1 function in different cellular contexts?

Several factors can contribute to variability in ZNRF1 functional studies across different experimental systems:

• Cell type-specific expression patterns: ZNRF1 expression varies across cell types and can be induced by specific stimuli such as poly(I:C), LPS, R848, and CpG . Basal expression levels should be quantified before functional studies.

• Compensatory mechanisms: Related E3 ligases may functionally compensate for ZNRF1 deficiency in certain cell types but not others, leading to context-dependent phenotypes.

• Stimulus-specific responses: ZNRF1's role differs depending on the pathway being activated. For example, ZNRF1 negatively regulates TLR3 responses but is dispensable for RLR-induced signaling .

• Post-translational regulation: ZNRF1 activity depends on its activation state, particularly phosphorylation by c-Src . Different cell types may exhibit varying levels of kinases that regulate ZNRF1.

• Experimental timing: Due to ZNRF1's role in receptor trafficking and degradation kinetics, the timing of measurements after stimulus application is critical for consistent results .

How can researchers confirm the specificity of ZNRF1 knockout or knockdown in their experimental systems?

To ensure specific and effective ZNRF1 depletion:

• Multi-level validation approach:

  • Genomic validation: Confirm CRISPR/Cas9 editing using PCR and sequencing of the targeted locus

  • Transcript analysis: Verify reduction in ZNRF1 mRNA using RT-qPCR with primers targeting multiple exons

  • Protein detection: Confirm absence of ZNRF1 protein via Western blotting with validated antibodies

• Functional validation: Assess known ZNRF1-dependent phenotypes:

  • Enhanced TLR3 signaling and increased type I IFN production upon poly(I:C) stimulation

  • Delayed EGFR degradation following EGF treatment

• Rescue experiments: Reintroduce wild-type ZNRF1 or functional mutants (e.g., C184A catalytic mutant) to verify that observed phenotypes are specifically due to ZNRF1 loss rather than off-target effects .

• Controls for compensation: Assess expression of related E3 ligases that might functionally compensate for ZNRF1 loss.

• Time-course studies: Evaluate the stability of knockdown or knockout over experimental duration, particularly for inducible or transient depletion systems.

How does ZNRF1 contribute to antiviral immunity and what methods best demonstrate this function?

ZNRF1 plays a critical role in regulating antiviral immunity, particularly against RNA viruses:

• Mechanism of action: ZNRF1 negatively regulates TLR3-mediated antiviral responses by promoting receptor degradation, thereby limiting type I interferon production .

• Viral infection models:

  • EMCV (Encephalomyocarditis virus): ZNRF1-deficient mice show enhanced resistance to EMCV infection with higher survival rates, increased serum IFN-β levels, and reduced viral replication in brain tissues .

  • SARS-CoV-2: Human lung epithelial cells lacking ZNRF1 express higher levels of type I interferons and produce less virus than control cells .

• Methodological approaches for studying ZNRF1 in viral infections:

  • In vivo infection models with ZNRF1 knockout mice

  • Measurement of viral replication using plaque assays or qPCR for viral genes

  • Assessment of type I interferon responses via ELISA or bioassays

  • Histopathological analysis of infected tissues to evaluate inflammation and tissue damage

  • Survival analysis to determine physiological significance

• Clinical relevance: ZNRF1 expression in peripheral blood mononuclear cells positively correlates with COVID-19 severity, suggesting potential prognostic value .

What experimental approaches are suitable for investigating ZNRF1's role in neural cell differentiation?

To study ZNRF1's functions in neural contexts:

• Neuronal culture systems:

  • Primary neuronal cultures from wild-type versus ZNRF1-deficient mice

  • Neural stem cell differentiation assays with ZNRF1 manipulation

  • Neuronal cell lines with ZNRF1 overexpression, which has been shown to cause neurite-like elongation

• Morphological analysis:

  • Quantification of neurite outgrowth parameters (length, branching, complexity)

  • Immunofluorescence microscopy to visualize neuronal markers and ZNRF1 localization

  • Live-cell imaging to monitor dynamic changes in neuronal morphology

• Molecular mechanisms:

  • Examination of ZNRF1's role in Wallerian degeneration through AKT degradation in response to oxidative stress

  • Investigation of synaptic vesicle membrane association in neurons

  • Proteomic identification of neuronal-specific ZNRF1 substrates

• Functional assays:

  • Electrophysiological recording to assess functional consequences of ZNRF1 manipulation

  • Neuronal activity mapping using calcium imaging or voltage-sensitive dyes

  • Behavioral testing in ZNRF1-deficient animal models to assess cognitive or motor phenotypes

What are the methodological considerations for analyzing contradictory roles of ZNRF1 in different immune pathways?

ZNRF1 exhibits apparently contradictory functions in different immune pathways, requiring careful experimental design to reconcile these roles:

• Pathway-specific analysis:

  • ZNRF1 negatively regulates TLR3-driven responses but is dispensable for RLR-induced signaling

  • ZNRF1 positively regulates TLR4-driven immune responses by mediating caveolin-1 ubiquitination and degradation, yet increases LPS-induced IRF3 activation

• Experimental strategies for dissecting dual roles:

  • Stimulus specificity: Compare responses to pathway-specific stimuli (poly(I:C) for TLR3, HMW/LMW poly(I:C) for MDA5/RIG-I, LPS for TLR4)

  • Temporal analysis: Examine early versus late responses to determine if ZNRF1 functions differently at various stages of immune activation

  • Substrate identification: Identify and validate pathway-specific ZNRF1 substrates (TLR3, caveolin-1) using proximity labeling or immunoprecipitation followed by mass spectrometry

• System-level approaches:

  • RNA sequencing to comprehensively analyze gene expression patterns in wild-type versus ZNRF1-deficient cells under different stimulation conditions

  • Phospho-proteomics to identify signaling nodes differentially regulated by ZNRF1 across immune pathways

  • Network modeling to integrate seemingly contradictory functions into a cohesive model of ZNRF1 action

• Physiological consequence assessment:

  • Dual infection models: Challenge with viral followed by bacterial infections to evaluate the consequences of enhanced antiviral but compromised antibacterial immunity in ZNRF1-deficient systems

  • Tissue-specific knockout models to determine if ZNRF1's functions vary across different immune cell populations