Alphascreen surefire p akt 1 2 3 p ser473 assay kit

[[(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 (MRS2365) was from Tocris (St. Louis, MO). N⁶-Cyclopentyladenosine (CPA), carbachol, PTX and 2-methylthioadenosine 5′-diphosphate trisodium salt (2MeSADP), adenosine-5′-N-ethyluronamide (NECA) were from Sigma (St. Louis, MO). UBO-QIC was purchased from University of Bonn (Germany). IP-One Tb HTRF kit was from Cisbio Bioassays (Bedford, MA). 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). HEK293 and DDT1-MF2 were from ATCC (Mannasas, VA); CHO cell lines stably expressing the human A₁AR, A_2AAR, and A_2BAR, and human M₃ and M₂ muscarinic acetylcholine receptors were made at the Laboratory of Bioorganic Chemistry, NIDDK (Bethesda, MD). 1321N1 astrocytoma cells expressing either the human P2Y₁R or P2Y₁₂R were from T. K. Harden (University of North Carolina, Chapel Hill, NC); all other reagents were from standard commercial sources and of analytical grade.

For the stimulation of ERK1/2 activity, the method used was as previously described [21 (link),22 (link)]. Briefly, CHO or 1321N1 astrocytoma cells (30,000 cells/100 μl) were seeded in a 96-well plate in complete growth medium. After cell attachment, medium was removed and cells were serum-starved overnight in medium without fetal bovine serum. Cells were pretreated with UBO-QIC (100 nM) or GO6983 (10 μM) for 30 min or PTX (200 ng/ml) overnight before the addition of agonists. Agonists were prepared in Hank's buffered salt solution, and cells were stimulated for 5 min. Medium was removed and cells were lysed with 1x Lysis Buffer (20 μl) (PerkinElmer AlphaScreen SureFire p-ERK1/2 (Thr202/Tyr204) Assay Kit) (PerkinElmer, Waltham, MA). Lysate (4 μl/well) was transferred to a 384-well ProxiPlate Plus (PerkinElmer). Reagents were added according the manual from the manufacturer, and the plate was measured using an EnVision multilabel reader using standard AlphaScreen settings. For the stimulation of Akt1/2/3 activity, the procedures were essentially the same as that of the ERK1/2 activity, except that the stimulation time for the P2Y₁ receptor is 20 min. The Akt activity was measured using AlphaScreen SureFire p-Akt 1/2/3 (p-Ser473) Assay Kit (PerkinElmer, Waltham, MA).