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Aberrant <jats:italic>NR2F1<\/jats:italic> causes the rare neurodevelopmental disorder Bosch-Boonstra-Schaaf Optic Atrophy Syndrome. In addition, altered <jats:italic>NR2F1<\/jats:italic> 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\u00a0role 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 <jats:italic>wild type<\/jats:italic> and <jats:italic>null<\/jats:italic> 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.<\/jats:p>","DOI":"10.1038\/s42003-025-07985-1","type":"journal-article","created":{"date-parts":[[2025,4,9]],"date-time":"2025-04-09T23:47:21Z","timestamp":1744242441000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Unravelling the conundrum of nucleolar NR2F1 localization using antibody-based approaches in vitro and in vivo"],"prefix":"10.1038","volume":"8","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4402-4974","authenticated-orcid":false,"given":"Michele","family":"Bertacchi","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Susanne","family":"Thei\u00df","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ayat","family":"Ahmed","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Eibl","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Agn\u00e8s","family":"Loubat","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gwendoline","family":"Maharaux","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wanchana","family":"Phromkrasae","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-6899-629X","authenticated-orcid":false,"given":"Krittalak","family":"Chakrabandhu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aylin","family":"Camg\u00f6z","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marco","family":"Antonaci","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christian Patrick","family":"Schaaf","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Miche\u0300le","family":"Studer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-6079-7017","authenticated-orcid":false,"given":"Magdalena","family":"Laugsch","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,4,10]]},"reference":[{"key":"7985_CR1","doi-asserted-by":"publisher","first-page":"1067","DOI":"10.1016\/j.jprot.2009.10.012","volume":"73","author":"C. 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