Surefire p erk1 2 thr202 tyr204 assay kit

Isoproterenol, formoterol, GO6983, GO6976, RO32–0432, GF109203X, H89, PSB603, ESI-09, TPPB, NECA (adenosine-5′-N-ethyluronamide), MRS5698 and MRS2365 ([[(1R,2R,3S,4R,5S)-4-[6-amino-2-(methylthio)-9H-purin-9-yl]-2,3-dihydroxybicyclo[3.1.0]hex-1-yl]methyl] diphosphoric acid monoester trisodium salt) were purchased from Tocris (Ellisville, MO). CGS21680">CGS21680, carbachol, cholera toxin (CTX) and PTX were from Sigma (St. Louis, MO). YM254890, enzastaurin, LY333531, VTX-27 and AB928 were purchased from MedChemExpress (Monmouth Junction, NJ, USA). BAY60-6583 (LUF6210, termed hereafter ‘BAY’) was synthesized at Leiden/Amsterdam Center for Drug Research and was provided by Ad IJzerman (Leiden, The Netherlands). All compounds were dissolved in DMSO except that CTX and PTX were in water, and proper controls were included in all experiments. AlphaScreen cAMP kit, SureFire p-ERK1/2 (Thr202/Tyr204) Assay Kit and AlphaScreen SureFire p-Akt 1/2/3 (p-Ser473) Assay Kit were purchased from PerkinElmer (Waltham, MA). G_s-null and G_q/11-null HEK293 cells were generated at Tohoku University, Sendai, Japan. HEK293 human embryonic kidney, PC-3 human prostate cancer, NIH-3T3 mouse fibroblast, and H9C2 rat cardiomyoblast cells were from ATCC (Manassas, VA); all other reagents were from standard commercial sources and of analytical grade.