A new phosphospecific cell-based ELISA for p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, protein kinase B and cAMP-response-element-binding protein.
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.
Versteeg
H H
HH
Laboratory for Experimental Internal Medicine, G2-130, Academic Medical Centre, Meibergdreef 9, NL-1105 AZ Amsterdam, The Netherlands.
Nijhuis
E
E
van den Brink
G R
GR
Evertzen
M
M
Pynaert
G N
GN
van Deventer
S J
SJ
Coffer
P J
PJ
Peppelenbosch
M P
MP
D000818:Animals; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt
D017362:Cyclic AMP Response Element-Binding Protein; D011518:Proto-Oncogene Proteins; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases