{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,8,26]],"date-time":"2025-08-26T06:23:44Z","timestamp":1756189424313},"reference-count":33,"publisher":"Wiley","issue":"4","license":[{"start":{"date-parts":[[2013,4,10]],"date-time":"2013-04-10T00:00:00Z","timestamp":1365552000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cytometry Part B Clinical"],"published-print":{"date-parts":[[2014,7]]},"abstract":"<jats:sec><jats:title>Background<\/jats:title><jats:p>Paradoxical activation of the MAP\u2010kinases, ERK1, and ERK2 (ERK1\/2) is observed in CML cell lines and primary CML patient cells treated with tyrosine kinase inhibitors (TKI) <jats:italic>in vitro<\/jats:italic>. The commonly accepted assumption is that activated ERK1\/2 is key regulators of survival of leukemic cells treated with kinase inhibitors. Hence, paradoxical ERK1\/2\u2010activation may trigger resistance <jats:italic>in vivo<\/jats:italic>, which yet has to be shown. We therefore sought to establish a flow cytometric assay that enables us to measure paradoxical TKI\u2010induced ERK1\/2\u2010activation on a single cell basis in primary CML cells.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>Side\u2010by\u2010side Western blot and intracellular flow cytometry (FCM) after <jats:italic>in vitro<\/jats:italic> exposure of cell lines and primary cells to nilotinib were performed. Detailed analysis of pre\u2010analytical factors and the issue of compartmentalization of phosphorylated ERK1\/2 by confocal laser scanning microscopy were performed.<\/jats:p><\/jats:sec><jats:sec><jats:title>Result<\/jats:title><jats:p>Results were conflicting in that pERK\u2010activation was robustly detected in Western blot assays, but not when cells were analyzed by FCM despite well functioning positive and negative controls. This is in contrast to experiments on other targets such as phospho\u2010CrkL, where also in our hands TKI\u2010dependent inhibition of phosphorylation is trackable by both Western blot and FCM assays.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p>To our knowledge this is the first report of discordant results in phospho\u2010protein analysis in TKI\u2010treated cells analyzed by Western blot vs. FCM. We speculate that a substance specific interaction interferes with fluorescence dependent methods seeking to track phosphorylated ERK1\/2 in TKI\u2010treated cells. \u00a9 2013 International Clinical Cytometry Society.<\/jats:p><\/jats:sec>","DOI":"10.1002\/cyto.b.21091","type":"journal-article","created":{"date-parts":[[2013,4,10]],"date-time":"2013-04-10T20:40:55Z","timestamp":1365626455000},"page":"229-235","source":"Crossref","is-referenced-by-count":6,"title":["Paradoxical MAPK\u2010activation in response to treatment with tyrosine kinase inhibitors in CML: Flow cytometry loses track"],"prefix":"10.1002","volume":"86","author":[{"given":"Ulf","family":"Schnetzke","sequence":"first","affiliation":[{"name":"Klinik f\u00fcr Innere Medizin II Abteilung f\u00fcr H\u00e4matologie und Internistische Onkologie Universit\u00e4tsklinikum Jena Erlanger Allee 101 07740 Jena 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