Simultaneous measurement of ERK, p38, and JNK MAP kinase cascades in vascular smooth muscle cells

Journal of Pharmacological and Toxicological Methods
D ChevalierBruce G Allen

Abstract

Activation of the mitogen-activated protein kinase (MAP kinase) pathways in cultured porcine aortic vascular smooth muscle cells (VSMCs) was determined following a 5-min stimulation with endothelin-1 (ET-1), phorbol 12-myristate 13-acetate (PMA), H2O2, or sodium arsenite. Extracellular signal-related kinase (ERK1/2), p38, and c-Jun N-terminal kinase (JNK1/2) MAP kinase activation was assessed using anti-phospho-MAPK kinase antibodies. The activation of these kinase cascades was also determined by resolving lysates on Mono Q using a fast protein liquid chromatography (FPLC) system and measuring the phosphorylation of specific substrates ERK1, c-Jun, and hsp27. The substrates were subsequently resolved from each other and the [gamma-32P]ATP in the reaction mixture by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and the incorporation of 32P was quantified by phosphor imaging. This technique revealed the presence of multiple peaks of activity phosphorylating ERK1 (5), c-Jun (7), and hsp27 (9). Differences in activation revealed by the chromatographic technique suggest that, although equivalent levels of activation may be detected by immunoblotting, the actual nature of the response differed depending upon the stimulus. Each st...Continue Reading

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Citations

Aug 11, 2012·Molecular and Cellular Biochemistry·Benoit BoivinBruce G Allen
Jun 17, 2003·Molecular and Cellular Biology·Marina Holgado-Madruga, Albert J Wong
Aug 1, 2006·International Journal of Experimental Pathology·Gordon FernsShahida Shafi
Feb 20, 2014·Journal of Cellular and Molecular Medicine·HaiYan ShanXiaoJuan Bai
Oct 16, 2002·Arteriosclerosis, Thrombosis, and Vascular Biology·Pedro GeraldesJean-François Tanguay
Nov 30, 2006·Journal of Cellular Biochemistry·Bingwen Jin, Yi-Ping Li
Jan 28, 2005·Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire·Hala Farhat, Bruce G Allen
Jul 12, 2002·Microscopy Research and Technique·Ronald G Tilton
Dec 5, 2002·Cellular Signalling·Benoit Boivin, Bruce G Allen

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