کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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1179833 | 962801 | 2013 | 6 صفحه PDF | دانلود رایگان |
We comparatively analyzed the basal activity of extra-cellular signal-regulated kinase (Erk1/2) in lysates of 10 human colorectal cancer cell lines by semi-quantitative Western blotting and time-resolved NMR spectroscopy. Both methods revealed heterogeneous levels of endogenous Erk1/2 activities in a highly consistent manner. Upon treatment with U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK) acting upstream of Erk1/2, Western-blotting and NMR congruently reported specific modulations of cellular phospho-Erk levels that translated into reduced kinase activities. Results obtained in this study highlight the complementary nature of antibody- and NMR-based phospho-detection techniques. They further exemplify the usefulness of time-resolved NMR measurements in providing fast and quantitative readouts of kinase activities and kinase inhibitor efficacies in native cellular environments. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).
► Direct, NMR-based detection of Erk1/2 kinase activity in cancer cell lysates.
► Quantitative measurements of Erk1/2 activity and inhibition by U0126.
► Comparison of semi-quantitative Western blotting and quantitative NMR readouts.
Journal: Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics - Volume 1834, Issue 7, July 2013, Pages 1396–1401