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Related Experiment Video

Updated: Aug 4, 2025

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay
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Issues with RNF43 antibodies to reliably detect intracellular location.

Shanshan Li1, Ruyi Zhang1, Marla Lavrijsen1

  • 1Department of Gastroenterology and Hepatology, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Rotterdam, The Netherlands.

Plos One
|April 6, 2023
PubMed
Summary

The E3 ubiquitin ligase RNF43 (Ring finger protein 43) is crucial for Wnt/β-catenin signaling. This study reveals common RNF43 antibodies are unreliable, suggesting nuclear localization is an artifact, not a true biological function.

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Area of Science:

  • Cellular Biology
  • Molecular Biology
  • Cancer Biology

Background:

  • RNF43 (Ring finger protein 43) negatively regulates Wnt/β-catenin signaling by internalizing Wnt receptors.
  • Mutations in RNF43 are implicated in various cancers, often linked to aberrant β-catenin signaling.
  • RNF43 has been proposed to have nuclear functions, influencing β-catenin signaling within the nucleus.

Purpose of the Study:

  • To critically evaluate the reliability of commonly used antibodies for detecting endogenous RNF43.
  • To investigate the proposed nuclear localization and function of RNF43.
  • To ensure accurate understanding of RNF43 biology for potential therapeutic applications.

Main Methods:

  • Generation of a RNF43-null cell line using genome editing (CRISPR/Cas9) to specifically delete exons 8 and 9.
  • Testing of four commercially available RNF43 antibodies using immunoblotting, immunofluorescence, and immunohistochemistry.
  • Comparative analysis of antibody signals in wild-type versus RNF43-null cells.

Main Results:

  • Four widely used RNF43 antibodies produced non-specific signals in all tested applications (immunoblotting, immunofluorescence, immunohistochemistry).
  • These antibodies failed to reliably detect endogenous RNF43 protein.
  • Observed nuclear staining patterns for RNF43 are likely artifacts caused by antibody cross-reactivity, not true RNF43 localization.

Conclusions:

  • Existing RNF43 antibodies are not suitable for reliably detecting endogenous RNF43 protein.
  • The presumed nuclear localization of RNF43 is an artifact of unreliable antibody detection.
  • Previous studies relying on these antibodies for RNF43 detection, particularly regarding nuclear functions, should be re-evaluated with caution.