title: 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.
abstract: 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.
authors.affiliation: Laboratory for Experimental Internal Medicine, G2-130, Academic Medical Centre, Meibergdreef 9, NL-1105 AZ Amsterdam, The Netherlands.
authors.forename: H H
authors.initials: HH
authors.lastname: Versteeg
authors.forename: E
authors.initials: E
authors.lastname: Nijhuis
authors.forename: G R
authors.initials: GR
authors.lastname: van den Brink
authors.forename: M
authors.initials: M
authors.lastname: Evertzen
authors.forename: G N
authors.initials: GN
authors.lastname: Pynaert
authors.forename: S J
authors.initials: SJ
authors.lastname: van Deventer
authors.forename: P J
authors.initials: PJ
authors.lastname: Coffer
authors.forename: M P
authors.initials: MP
authors.lastname: Peppelenbosch
mesh_terms: 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
chemical_list: 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