MK-0752, a γ-secretase inhibitor, reduces Aβ40 production(IC50=5 nM).

β-Amyloid:5 nM

Protein kinase assays : Protein kinase assays are either done in-house by ELISA-based assay methods (Kit, KDR, PDGFRα, and PDGFRβ) or by a radiometric method. In-house ELISA assays used poly(Glu:Tyr) as the substrate bound to the surface of 96-well assay plates; phosphorylation is then detected using an antiphosphotyrosine antibody conjugated to HRP. The bound antibody is then quantitated using ABTS as the peroxidase substrate by measuring the absorbance at 405/490 nm. All assays uses purified recombinant kinase catalytic domains that are either expressed in insect cells or in bacteria. The Kit and EGFR protein used for in-house assays are prepared internally; other enzymes are obtained. Recombinant Kit protein is expressed as an NH2-terminal glutathione S-transferase fusion protein in insect cells and is initially purified as a nonphosphorylated (nonactivated) enzyme with a relatively high Km for ATP (400 μM). In some assays, an activated (tyrosine phosphorylated) form of the enzyme is prepared by incubation with 1 mM ATP for 1 hour at 30 °C. The phosphorylated protein is then passed through a desalting column to remove the majority of the ATP and stored at ?80 °C in buffer containing 50% glycerol. The resultant preparation has a considerably higher specific activity and a lower Km for ATP (25 μM) than the initial nonphosphorylated preparation. The inhibition of Kit autophosphorylation by OSI-930 is assayed by incubation of the nonphosphorylated enzyme at 30 °C in the presence of 200 μM ATP and various concentrations of OSI-930. The reaction is stopped by removal of aliquots into SDS-PAGE sample buffer followed by heating to 100 °C for 5 minutes. The degree of phosphorylation of Kit is then determined by immunoblotting for both total Kit and phosphorylated Kit.

H2O: < 1 mg/mL (insoluble or slightly soluble)

DMSO: 140 mg/mL (316.1 mM), Sonication is recommended.

Ethanol: 45 mg/mL (101.6 mM), Sonication is recommended.

10% DMSO+40% PEG300+5% Tween 80+45% Saline: 3.3 mg/mL (7.45 mM), Sonication is recommended.