Abstract
Extracellular signal-regulated kinase 5 (ERK5), also known as big MAPK (BMK1), is the most recently identified member of the mitogen-activated kinase pathway. It is ubiquitously expressed in mammalian cells and is activated by a number of growth factors. Gene knockout studies in mice have shown a critical role for ERK5 cardiovascular development and vascular integrity. Current methods to detect ERK5 activation in cells have relied on in vitro kinase assays and more recently phospho-specific antibodies. However, antibodies produced against phosphorylated proteins can often yield inconsistent data. Phos-tag™ Acrylamide is a reagent that enables specific tagging of phosphorylated proteins, resulting in retarded mobility and a distinct upward band shift from the non-phosphorylated protein following SDS-PAGE. Here, we describe the details of Phosphate affinity SDS-PAGE of ERK5 using acrylamide-pendant Phos-tag™.
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Acknowledgements
The authors would like to thank the Biotechnology and Biological Sciences Research Council (BBSRC) for funding research on ERK5 in our group.
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Nithianandarajah-Jones, G.N., Cross, M.J. (2015). Analysis of VEGF-Mediated ERK5 Activity in Endothelial Cells. In: Fiedler, L. (eds) VEGF Signaling. Methods in Molecular Biology, vol 1332. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2917-7_9
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DOI: https://doi.org/10.1007/978-1-4939-2917-7_9
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2916-0
Online ISBN: 978-1-4939-2917-7
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