Analysis and Regulation of Vasodilator-stimulated Phosphoprotein Serine 239 Phosphorylation in Vitro and in Intact Cells Using a Phosphospecific Monoclonal Antibody
作者： Albert Smolenski , Christiane Bachmann , Kathrin Reinhard , Petra Hönig-Liedl , Thomas Jarchau
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摘要: The development and functional analysis of a monoclonal antibody (16C2) are reported; the recognizes vasodilator-stimulated phosphoprotein (VASP; an established substrate both cAMP- cGMP-dependent protein kinase) only when serine 239 is phosphorylated. VASP represents one best characterized kinase phosphorylation sites in vitro intact cells. Experiments with purified, recombinant human various constructs mutated (S157A, S239A, T278A) experiments cells (human/rat platelets other cells) treated cyclic nucleotide-elevating agents demonstrated specificity 16C2. Quantitative shift from 46 to 50 kDa (indicating 157 phosphorylation) appearance detected by 16C2 (VASP stimulated selective activators confirmed that site preferred Immunofluorescence cGMP analogs showed also detects situ at intracellular localization sites. Analysis appears be method presently available measure activation Also, promises excellent tool for evaluation function
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