scholarly journals Neurotransmitter- and Growth Factor-Induced cAMP Response Element Binding Protein Phosphorylation in Glial Cell Progenitors: Role of Calcium Ions, Protein Kinase C, and Mitogen-Activated Protein Kinase/Ribosomal S6 Kinase Pathway

1997 ◽  
Vol 17 (4) ◽  
pp. 1291-1301 ◽  
Author(s):  
Mario Pende ◽  
Tracey L. Fisher ◽  
Peter B. Simpson ◽  
James T. Russell ◽  
John Blenis ◽  
...  
2000 ◽  
Vol 350 (3) ◽  
pp. 717-722 ◽  
Author(s):  
Henri H. VERSTEEG ◽  
Evert NIJHUIS ◽  
Gijs R. VAN DEN BRINK ◽  
Maaike EVERTZEN ◽  
Gwenda N. PYNAERT ◽  
...  

Assaying activation of signal transduction is laborious and does not allow the study of large numbers of samples, essential for high-throughput drug screens or for large groups of patients. Using phosphospecific antibodies, we have developed ELISA techniques enabling non-radioactive semi-quantitative assessment of the activation state of p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, protein kinase B and the transcription factor cAMP-response-element-binding protein (CREB) in 96-well plates. This assay has been termed PACE (phosphospecific antibody cell-based ELISA) and was used successfully for both adherent and suspension cells. Various stimuli induced dose-dependent enzymic activity of which the kinetics closely correlated with those measured via classical methodology. Using PACE we have now characterized for the first time the concentration-dependent effects of various inflammatory prostaglandins on CREB phosphorylation in macrophages. PACE is a straightforward and novel technique enabling the large-scale analysis of signal transduction.


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