RNF43 Antibody, FITC conjugated

What is RNF43 and what cellular functions does it regulate?

RNF43 (Ring Finger Protein 43) functions as an E3 ubiquitin-protein ligase that acts as a negative regulator of the Wnt signaling pathway. It mediates the ubiquitination, endocytosis, and subsequent degradation of Wnt receptor complex components, particularly Frizzled (FZD) receptors. RNF43 functions in both canonical and non-canonical Wnt signaling pathways . As a tumor suppressor in the intestinal stem cell zone, RNF43 inhibits Wnt signaling, thereby restricting the size of the intestinal stem cell zone . Recent research has also demonstrated that RNF43 inhibits WNT5A-driven signaling and can suppress melanoma progression . Understanding RNF43's cellular localization and interactions is critical for research into Wnt pathway dysregulation in cancer and developmental disorders.

What are the key specifications of commercially available RNF43 antibodies with FITC conjugation?

The FITC-conjugated RNF43 antibody (catalog ABIN7151200) recognizes amino acids 566-697 of human RNF43 . This rabbit polyclonal antibody has the following specifications:

The antibody is highly purified (>95%) using Protein G purification techniques and is suitable for various experimental applications requiring fluorescent detection of RNF43 .

How does RNF43 interact with the Wnt signaling pathway components?

RNF43 interacts with multiple components of both canonical and non-canonical Wnt signaling pathways. For the canonical pathway, RNF43 primarily mediates the ubiquitination of Frizzled receptors, leading to their endocytosis and degradation . Recent research has revealed specific preferences in these interactions - RNF43 preferentially down-regulates FZD1, FZD5, and FZD7, whereas the related E3 ligase ZNRF3 displays a preference toward FZD6 .

In the non-canonical pathway, RNF43 interacts with ROR1/ROR2 and VANGL1/VANGL2 proteins in a DVL-independent manner . Specifically, co-immunoprecipitation experiments have demonstrated that RNF43 can efficiently pull down these proteins, and deletion of the cysteine-rich domain (CRD) in ROR2 does not impact this interaction, suggesting that RNF43 primarily interacts with RORs intracellularly . Furthermore, RNF43 can ubiquitinate VANGL2 and DVL proteins, acting as a negative regulator of non-canonical Wnt signaling .

What are the optimal protocols for using FITC-conjugated RNF43 antibodies in immunofluorescence experiments?

When using FITC-conjugated RNF43 antibodies for immunofluorescence experiments, researchers should follow these methodological considerations:

• Sample preparation: Fix cells or tissues appropriately (4% paraformaldehyde for 15-20 minutes at room temperature is standard). For tissues, consider antigen retrieval methods if necessary.

• Blocking: Use 5-10% normal serum (matching the secondary antibody host) with 0.1-0.3% Triton X-100 for permeabilization in PBS for 1 hour at room temperature.

• Antibody dilution: For the FITC-conjugated RNF43 antibody targeting AA 566-697, optimal dilutions should be determined empirically, starting with a range of 1:50-1:200 as suggested for similar applications with unconjugated RNF43 antibodies .

• Incubation conditions: Incubate overnight at 4°C in a humidified chamber to ensure optimal binding.

• Controls: Include both positive controls (tissues known to express RNF43) and negative controls (secondary antibody only, or non-specific IgG) to validate specificity.

• Counterstaining: Use DAPI (1:1000) for nuclear visualization, and consider phalloidin for F-actin if cellular morphology is important.

• Mounting: Use anti-fade mounting medium specific for fluorescent preservation.

• Imaging considerations: Use appropriate excitation/emission filters for FITC (excitation ~495nm, emission ~520nm) and avoid photobleaching by minimizing exposure to light.

For co-localization studies with RNF43 and its binding partners (ROR1/ROR2, VANGL1/VANGL2), researchers have successfully demonstrated co-localization at the plasma membrane, suggesting these are important sites to examine .

How can researchers validate the specificity of RNF43 antibodies in their experimental systems?

Validating antibody specificity is crucial for ensuring reliable results with RNF43 antibodies. Researchers should employ multiple approaches:

• Western blotting: Compare band patterns with expected molecular weight (~83-90 kDa for full-length RNF43). Unconjugated variants of RNF43 antibodies have been validated for Western blotting at dilutions of 1:300-5000 .

• RNF43 knockdown/knockout controls: Use siRNA, shRNA, or CRISPR-Cas9 systems to reduce or eliminate RNF43 expression, then confirm reduced antibody signal.

• Overexpression validation: Transfect cells with RNF43-expressing constructs and confirm increased antibody signal.

• Multiple antibodies targeting different epitopes: Compare staining patterns using antibodies recognizing different regions of RNF43 (e.g., AA 41-140, AA 543-572, AA 566-697, AA 504-553) .

• Immunoprecipitation followed by mass spectrometry: Confirm antibody pulls down RNF43 protein by identifying peptides via mass spectrometry.

• Cross-reactivity testing: If working with non-human models, confirm species-specific reactivity. The FITC-conjugated antibody (AA 566-697) is reactive with human samples, while other RNF43 antibodies may react with mouse, rat, cow, dog, horse, or rabbit samples .

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide to block specific binding sites before staining.

What are the best experimental approaches for studying RNF43-mediated ubiquitination of target proteins?

Studying RNF43-mediated ubiquitination requires specialized experimental approaches:

• His-ubiquitin pulldown assay: This method has been successfully used to demonstrate that VANGL2, DVL1, and DVL2 are ubiquitinated when co-expressed with RNF43 but not with enzymatically inactive RNF43 Mut1 variant . Researchers should:

  • Co-express His-tagged ubiquitin with RNF43 and the target protein

  • Lyse cells under denaturing conditions to disrupt non-covalent interactions

  • Perform Ni-NTA pulldown to isolate His-ubiquitin conjugated proteins

  • Analyze by immunoblotting with antibodies against the target protein

• Co-immunoprecipitation with ubiquitin antibodies: Immunoprecipitate the target protein and blot for ubiquitin, or vice versa.

• Proteasome inhibition: Treat cells with MG132 or other proteasome inhibitors to prevent degradation of ubiquitinated proteins, enhancing detection.

• Comparison with enzymatically inactive mutants: Use the RNF43 Mut1 variant as a negative control, as it has been shown to abolish ubiquitination activity .

• In vitro ubiquitination assays: Reconstitute the ubiquitination reaction using purified components (E1, E2, RNF43, substrate, ubiquitin, ATP) to determine direct enzymatic activity.

• Mass spectrometry analysis: Identify specific lysine residues modified by ubiquitin to map the ubiquitination sites on target proteins.

• Proximity-dependent biotin identification (BioID): This technique has been used to identify novel protein-protein interactions of RNF43 with components of the non-canonical Wnt pathway .

How does the transmembrane domain of RNF43 influence its specificity for different Frizzled receptors?

Recent research has revealed that the transmembrane domain (TMD) of RNF43 is a key molecular determinant for its specificity toward certain Frizzled receptors. RNF43 preferentially down-regulates FZD1, FZD5, and FZD7, whereas the related E3 ligase ZNRF3 displays a preference toward FZD6 .

Mechanistically, the TMD appears to be crucial for inducing FZD5 endocytosis. Experimental evidence shows that a TMD swap between RNF43 and ZNRF3 re-directs their preference for FZD5 down-regulation, confirming the critical role of this domain in determining receptor specificity . This functional specificity correlates with tissue-specific expression patterns of FZD homologues and the incidence of RNF43 or ZNRF3 mutations in different cancer types.

Researchers investigating this aspect should consider:

• Using chimeric constructs where TMDs are swapped between RNF43 and ZNRF3

• Employing pulse-chase experiments with SNAP-tagged FZD receptors to quantify internalization rates

• Utilizing flow cytometry to measure surface levels of different FZD receptors upon co-expression with wild-type or chimeric E3 ligases

• Conducting structural studies to determine the specific TMD residues that mediate these interactions

What role does RNF43 play in cancer progression, and how can RNF43 antibodies be used in cancer research?

RNF43 functions as a tumor suppressor by negatively regulating Wnt signaling, which is frequently dysregulated in cancer. Research has demonstrated that RNF43 inhibits WNT5A-driven signaling and suppresses melanoma progression . Understanding its role in different cancer types requires sophisticated research approaches using RNF43 antibodies:

• Tissue microarray analysis: Use RNF43 antibodies for immunohistochemistry (IHC-P, 1:200-400 dilution) to evaluate expression patterns across cancer types and correlate with patient outcomes.

• Mutation analysis correlation: Compare RNF43 protein expression/localization with mutation status to understand genotype-phenotype relationships. The tissue-specific expression patterns of FZD homologues correlate with the incidence of RNF43 or ZNRF3 mutations across different cancer types .

• Functional studies in cancer models: Use RNF43 antibodies to validate knockout/knockdown efficiency in cancer cell lines or patient-derived xenografts when studying effects on:

  • Cell proliferation and survival

  • Migration and invasion

  • Response to Wnt pathway modulators

  • Tumor growth in vivo

• Signaling pathway analysis: Use phospho-specific antibodies alongside RNF43 antibodies to map how RNF43 deficiency affects downstream signaling cascades.

• Biomarker development: Evaluate RNF43 as a potential biomarker for cancer prognostication or treatment selection, particularly in cancers with Wnt pathway dysregulation.

• Drug resistance mechanisms: Investigate how RNF43 mutations or expression changes contribute to resistance to Wnt pathway inhibitors or other targeted therapies.

How does the DVL-independent interaction of RNF43 with PCP proteins differ from its canonical pathway interactions?

Recent research has uncovered that RNF43 interacts with planar cell polarity (PCP) proteins from the VANGL and ROR families in a DVL-independent manner, distinguishing these interactions from its canonical Wnt pathway functions . These findings represent an important advance in understanding RNF43's broader roles in cell signaling.

Key differences include:

• Interaction mechanisms: While DVL proteins mediate ubiquitination of FZD receptors by RNF43 in the canonical Wnt/β-catenin pathway , co-immunoprecipitation experiments in DVL triple knockout cells demonstrate that RNF43 can bind both VANGL2 and ROR1 as efficiently as in wild-type cells . This indicates a fundamentally different interaction mechanism for these PCP proteins.

• Subcellular localization: Co-localization experiments show that VANGL2 and RNF43 interact at the cell membrane . Similarly, both ROR1/ROR2 co-localize with RNF43 at the plasma membrane . This localization is important for understanding the spatial regulation of these interactions.

• Domain requirements: For ROR interactions, deletion of the cysteine-rich domain (CRD) has no impact on the amount of co-immunoprecipitated RNF43, suggesting RNF43 primarily interacts with RORs intracellularly through different domains than those used for canonical pathway interactions .

• Functional outcomes: While RNF43 mediates endocytosis and degradation of FZD receptors in the canonical pathway, its interactions with VANGL and ROR proteins appear to regulate non-canonical Wnt signaling through ubiquitination of these partners, thereby affecting different cellular processes like cell polarity and migration .

Researchers studying these interactions should use co-immunoprecipitation assays, subcellular fractionation, and advanced microscopy techniques with appropriate antibodies to further characterize these protein-protein interactions.

What are common pitfalls when using FITC-conjugated antibodies, and how can researchers overcome them?

When working with FITC-conjugated RNF43 antibodies, researchers should be aware of several common technical challenges:

• Photobleaching: FITC is prone to photobleaching under prolonged exposure to excitation light.

  • Solution: Use anti-fade mounting media, minimize exposure time during imaging, consider using more photostable fluorophores for extended imaging sessions.

• Autofluorescence: Tissue samples often exhibit autofluorescence in the FITC emission range, particularly formalin-fixed tissues.

  • Solution: Include unstained control samples, use autofluorescence quenching agents (e.g., Sudan Black B, TrueBlack), or employ spectral unmixing during image acquisition.

• pH sensitivity: FITC fluorescence is sensitive to pH changes, with optimal performance at pH 8.0.

  • Solution: Carefully control buffer pH during staining and washing steps, particularly when studying membrane proteins like RNF43 that traverse cellular compartments with different pH environments.

• Signal-to-noise ratio: Direct conjugation eliminates the amplification step of secondary antibodies.

  • Solution: Optimize antibody concentration carefully, consider tyramide signal amplification (TSA) for low-abundance targets.

• Fixation artifacts: Improper fixation can mask epitopes or create false-positive signals.

  • Solution: Compare multiple fixation methods (paraformaldehyde, methanol, acetone) to determine optimal protocol for RNF43 detection.

• Storage conditions: FITC conjugates have limited stability.

  • Solution: Store at -20°C protected from light, avoid repeated freeze-thaw cycles, prepare working aliquots, and follow manufacturer recommendations for the specific conjugate .

How can researchers optimize detection of RNF43 in different subcellular compartments?

RNF43 is reported to localize to multiple subcellular compartments including the cytoplasm, nucleus, and cell membrane . Optimizing detection across these compartments requires specific considerations:

• Membrane localization:

  • Use gentle permeabilization (0.1% Triton X-100 or 0.01% saponin)

  • Consider membrane co-markers (Na⁺/K⁺-ATPase, E-cadherin) for co-localization studies

  • When studying RNF43 interactions with membrane proteins like VANGL2 or ROR1/ROR2, optimize fixation to preserve membrane structure

• Nuclear localization:

  • Ensure adequate nuclear permeabilization (0.3-0.5% Triton X-100)

  • Consider antigen retrieval methods for formalin-fixed samples

  • Use nuclear counterstains (DAPI, Hoechst) for precise localization

  • Include nuclear extraction controls when performing Western blots

• Cytoplasmic localization:

  • Balance permeabilization to allow antibody access without extracting soluble proteins

  • Consider cytoskeletal preservation with simultaneous paraformaldehyde/glutaraldehyde fixation

• Endosomal compartments:

  • Since RNF43 mediates endocytosis of Wnt receptor complex components, co-staining with endosomal markers (Rab5, Rab7, EEA1) may be informative

  • Consider live-cell imaging with pH-sensitive fluorophores to track receptor internalization

• Technical approach optimizations:

  • For subcellular fractionation: Validate fraction purity with compartment-specific markers

  • For super-resolution microscopy: Use appropriate mounting media and sample preparation techniques

  • For biochemical assays: Adjust lysis conditions to effectively extract RNF43 from all compartments

What are the most effective controls for validating RNF43 antibody specificity in complex experimental designs?

For complex experimental designs involving RNF43 antibodies, particularly FITC-conjugated variants, implementing rigorous controls is essential:

• Genetic controls:

  • CRISPR/Cas9 knockout of RNF43 provides the most definitive negative control

  • siRNA knockdown with multiple siRNAs targeting different regions of RNF43 mRNA

  • Rescue experiments with RNF43 expression constructs in knockout backgrounds

  • Use of mutant variants like the enzymatically inactive RNF43 Mut1 for functional studies

• Antibody validation controls:

  • Peptide competition assays using the immunizing peptide (AA 566-697 for the FITC-conjugated antibody)

  • Comparison of staining patterns with multiple antibodies targeting different epitopes of RNF43

  • Isotype control (rabbit IgG-FITC) at matching concentration

  • Titration series to determine optimal antibody concentration

  • Secondary-only controls when using unconjugated primary antibodies

• Expression verification controls:

  • Parallel validation with mRNA detection methods (qRT-PCR, RNAscope)

  • Correlation with proteomic data when available

  • Comparison with GFP-tagged RNF43 in transfection experiments

• Application-specific controls:

  • For FZD down-regulation studies: FZD5 or FZD6 expression levels in cells with/without RNF43

  • For ubiquitination assays: Include both wild-type RNF43 and enzymatically inactive RNF43 Mut1

  • For co-IP experiments: Include both RNF43 and control protein to verify specific interactions

  • For subcellular localization: Include known RNF43 interactors (VANGL2, ROR1) as positive co-localization controls

By implementing these comprehensive controls, researchers can ensure the validity of their findings when studying RNF43 using FITC-conjugated antibodies across diverse experimental contexts.

How might new developments in antibody technology enhance RNF43 research?

Emerging antibody technologies offer promising opportunities to advance RNF43 research:

What are the implications of RNF43's differential specificity for FZD receptors in therapeutic development?

The discovery that RNF43 preferentially down-regulates specific FZD receptors (FZD1/FZD5/FZD7) while ZNRF3 favors others (e.g., FZD6) has significant implications for therapeutic development:

• Targeted therapeutic approaches: Understanding receptor specificity enables more precise targeting of Wnt signaling in cancers with specific FZD receptor expression patterns. Therapies could be tailored based on the predominant FZD receptor subtype in a particular cancer.

• Predictive biomarkers: The relationship between tissue-specific expression patterns of FZD homologues and the incidence of RNF43 or ZNRF3 mutations suggests potential biomarkers for predicting response to Wnt pathway inhibitors.

• Synthetic ligases: Engineering RNF43 variants with altered specificity could create therapeutic tools that selectively target specific FZD receptors for degradation.

• Transmembrane domain mimetics: Since the TMD is a key determinant for inducing FZD5 endocytosis , developing peptides or small molecules that mimic these interactions could lead to novel therapeutic agents.

• Combination therapies: Different cancers may require targeting of multiple FZD receptors based on their expression profiles. Understanding which E3 ligases target which receptors enables more rational combination approaches.

• Resistance mechanisms: The differential specificity explains why mutations in either RNF43 or ZNRF3 may have distinct phenotypic consequences and suggests potential resistance mechanisms to Wnt pathway inhibitors.

• Developmental implications: Beyond cancer, this specificity may explain the distinct developmental roles of RNF43 and ZNRF3, informing regenerative medicine approaches that modulate the Wnt pathway.

Research tools like FITC-conjugated RNF43 antibodies will be essential for investigating these therapeutic implications in preclinical models and patient samples.