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在酪氨酸激酶抑制剂（TKI）治疗初发慢性粒细胞白血病（CML）患者时，可出现MAP激酶、ERK1、ERK2的矛盾性活化，这通常认为可能是CML细胞耐药的机制之一。在本文中，作者同时利用Western blot和流式检测了TKI处理的细胞株和TKI治疗的患者细胞，发现利用WESTERN BLOT检测到pERK1/2明显的活化，但流式却检测不到。作者推测可能是由于TKI处理过的细胞中，存在某种未知的物质干扰了荧光素的结合，而对无荧光素的Western blot无影响。（流式中文网翻译）
原文免费在线阅读和下载参见本帖2楼。
western blot和流式的矛盾结果
＝＝＝＝＝＝原文摘要，感谢EthanMr站友的无私分享，奖励流星2颗＝＝＝＝＝＝＝
Cytometry B Clin Cytom. 2013 Apr 10. doi: 10.1002/cyto.b.21091. [Epub ahead of print]
Paradoxical MAPK-activation in response to treatment with tyrosine kinase inhibitors in CML: Flow cytometry loses track.
Schnetzke U , Fischer M , Frietsch JJ , Finkensieper A , Clement JH , Hochhaus A , Rosée PL .
Source
Klinik für Innere Medizin II, Abteilung für Hämatologie und Internistische Onkologie, Universitätsklinikum Jena, Erlanger Allee 101, 07740 Jena, Germany.
Abstract
BACKGROUND:
Paradoxical activation of the MAP-kinases, ERK1, and ERK2 (ERK1/2) is observed in CML cell lines and primary CML patient cells treated with tyrosine kinase inhibitors (TKI) in vitro. 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-activation may trigger resistance in vivo, which yet has to be shown. We therefore sought to establish a flow cytometric assay that enables us to measure paradoxical TKI-induced ERK1/2-activation on a single cell basis in primary CML cells.
METHODS:
Side-by-side Western blot and intracellular flow cytometry (FCM) after in vitro exposure of cell lines and primary cells to nilotinib were performed. Detailed analysis of pre-analytical factors and the issue of compartmentalization of phosphorylated ERK1/2 by confocal laser scanning microscopy were performed.
RESULT:
Results were conflicting in that pERK-activation 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-CrkL, where also in our hands TKI-dependent inhibition of phosphorylation is trackable by both Western blot and FCM assays.
CONCLUSIONS:
To our knowledge this is the first report of discordant results in phospho-protein analysis in TKI-treated 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-treated cells.
PMID: 23576291
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发表于 2013-4-12 21:59:51
发表于 2013-4-19 14:02:03
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