Analysis of VEGF-Mediated ERK5 Activity in Endothelial Cells.
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™.
Department of Molecular and Clinical Pharmacology, MRC Centre for Drug Safety Science, Institute of Translational Medicine, University of Liverpool, Crown Street, Liverpool, L69 3GE, UK.
Gopika N
GN
Nithianandarajah-Jones
Michael J
MJ
Cross
D015153:Blotting, Western; D042783:Endothelial Cells / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D048054:Mitogen-Activated Protein Kinase 7 / Q000378:metabolism*; D042442:Vascular Endothelial Growth Factors / Q000378:metabolism*
D042442:Vascular Endothelial Growth Factors; D048054:Mitogen-Activated Protein Kinase 7