# Unravelling the conundrum of nucleolar NR2F1 localization using antibody-based approaches in vitro and in vivo

## TL;DR

This paper investigates the localization of the NR2F1 protein in the nucleolus and finds that its presence there is an artifact, not a functional role.

## Contribution

The study reveals that nucleolar NR2F1 localization is artificial and identifies optimal antibodies for accurate localization studies.

## Key findings

- NR2F1 nucleolar localization is an artifact, not a functional role.
- Seven anti-NR2F1 antibodies were tested to determine their suitability for in vitro and in vivo studies.
- The study emphasizes the importance of antibody optimization for accurate protein localization.

## Abstract

As a transcription factor, NR2F1 regulates spatiotemporal gene expression in the nucleus particularly during development. Aberrant NR2F1 causes the rare neurodevelopmental disorder Bosch-Boonstra-Schaaf Optic Atrophy Syndrome. In addition, altered NR2F1 expression is frequently observed in various cancers and is considered a prognostic marker or potential therapeutic target. NR2F1 has been found in both the nucleus and nucleoli, suggesting a non-canonical and direct role in the latter compartment. Hence, we studied this phenomenon employing various in vitro and in vivo models using different antibody-dependent approaches. Examination of seven commonly used anti-NR2F1 antibodies in different human cancer and stem cells as well as in wild type and null mice revealed that NR2F1 nucleolar localization is artificial and has no functional role. Our subsequent comparative analysis demonstrated which anti-NR2F1 antibody best fits which approach. The data allow for correct data interpretation and underline the need to optimize any antibody-mediated technique.

Antibody optimization is crucial for proper data interpretation, such as determining accurate protein localization. Here, this is performed for NR2F1, a transcription factors associated with a rare neurodevelopmental disorder and cancer.