Kinase experiment: | Before the scintillation proximity assay (SPA) assay, 200 nM recombinant AMPK protein (α1β1γ1, α2β1γ1, α1β2γ1, α2β2γ1, α1(1-394), α1(1-335), α1(1-312)) is constructed, expressed, purified and fully phosphorylated. The SPA reactions are performed in 96-well plates in a final volume of 50 µL containing 20 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 1 mM DTT, 2 µM biotin-SAMS, 2 µM ATP and 7.4×103 Bq/well [γ-33P]ATP. The reactions are initiated by the addition of 50 nM recombinant AMPK protein to the reaction solutions, followed by incubation at 30°C for 2 hr. The reactions are then terminated by the addition of 40 µL of stop solution containing 80 µg Streptavidin-coated SPA beads per well, 50 mM EDTA and 0.1% Triton X-100 in PBS, pH 7.5, followed by incubation for 1 hr. Finally, 160 µL of suspension solution containing 2.4 M CsCl, 50 mM EDTA and 0.1% Triton X-100 in PBS, pH 7.5, is added to the reaction solution to suspend the SPA beads completely. The SPA signals are measured in a Wallac Microbeta plate counter 30 min later[1]. |
Animal experiment: | Mice[1]C57BKS db/db mice are maintained under a 12 hr light-dark cycle with free access to water and food. At 8 weeks of age, male db/db mice are randomly assigned to the various treatment groups by body weight and glucose levels (n=6-8). The treatment groups for the 5-week chronic study are as follows: vehicle (0.5% methylcellulose), ZLN024 (15 mg/kg) and Metformin (250 mg/kg). The treatments are orally administered once daily. The body weights and food intake are measured daily. After 5 weeks of treatment, the mice are killed after a final dose, and the tissues are collected for further analysis. |
