Unravelling the conundrum of nucleolar NR2F1 localization: A comparative analysis of NR2F1 antibody-based approachesin vitroandin vivo

ABSTRACT As a transcription factor, NR2F1 regulates spatiotemporal gene expression during development and in adulthood. Aberrant NR2F1 causes a rare neurodevelopmental disorder known as 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. In this context, NR2F1 has been shown to localize not only in the nucleus but also in the nucleoli, suggesting a novel non-canonical role in this compartment. Hence, we studied this phenomenon employing various in vitro and in vivo models in 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 the nucleolar localization of NR2F1 is artificial and does not play a functional role. Our subsequent comparative analysis demonstrated for the first time which anti-NR2F1 antibody best fits which approach. As our data allow for correct data interpretation, making them publicly available may have far-reaching implications for NR2F1 research in health and disease. More generally, the study also underlines the need to optimize any antibody-mediated technique.