Development of EIA systems for active-form MAP kinase.
Quantitative sandwich enzyme immunoassay (EIA) systems, that can distinguish between active-form subtypes of mitogen-activated protein kinases (p44 and p42 MAP kinase, also called ERK1 and ERK2), were developed employing subtype-specific antibodies as a solid phase and an antibody specific for the phosphorylated region of MAP kinases as the detector. Using these systems, we investigated the dynamic changes in the activity of ERK1 and ERK2 in platelet-derived growth factor (PDGF)-treated rat mesangial cells and nerve growth factor (NGF)-treated PC12. Both ERK1 and ERK2 were activated immediately after stimulation, and the activity reached a maximum at 5-10 min. The total activity of both subtypes correlated well with that obtained using the conventional method. Compared with the usual methods, these systems should have a higher specificity and be more convenient and suitable for experiments with multiple samples. Moreover, as these EIA systems can be applied not only to rat MAP kinases but also to human, mouse and rabbit MAP kinases, they are potentially very useful for a range of investigations.
Tani
A
A
Discovery Research Laboratories III, Takeda Chemical Industries Ltd., 2-17-85 Juso-honmachi, Yodogawa-ku, Osaka, Japan. tani_akiyoshi@takeda.co.jp
Noda
M
M
Ichimori
Y
Y
D000804:Angiotensin II / Q000494:pharmacology; D057911:Angiotensin Receptor Antagonists; D000818:Animals; D000918:Antibody Specificity; D001562:Benzimidazoles / Q000494:pharmacology; D001713:Biphenyl Compounds; D002478:Cells, Cultured; D004789:Enzyme Activation; D005920:Glomerular Mesangium / Q000166:cytology; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D008297:Male; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000032:analysis* / Q000276:immunology; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000032:analysis* / Q000276:immunology; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D016716:PC12 Cells; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D013777:Tetrazoles / Q000494:pharmacology
D057911:Angiotensin Receptor Antagonists; D001562:Benzimidazoles; D001713:Biphenyl Compounds; D013777:Tetrazoles; D000804:Angiotensin II; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C081643:candesartan