kinase assays: PI3K inhibition by LY294002 is determined in a radiometric assay using purified, recombinant enzymes with 1?μM ATP. The kinase reaction is carried out for 1?hour at room temperature (24oC) and is terminated by addition of PBS. IC50 values are subsequently determined using a sigmoidal dose–response curve fit (variable slope). CK2 and GSK3β (glycogen synthase kinase 3β) inhibition is established by kinase selectivity screening. LY294002 is tested against the Upstate panel of kinases in 10?μM ATP.
SB-3CT is dissolved in DMSO (10 mM) and stored (-22°C), and then diluted with appropriate media (DMSO 1%) before use. PC3 cells are seeded in 35-mm dishes (5×104 cells/dish) in complete culture medium. The next day, the medium is replaced with complete medium supplemented with 1% DMSO alone (vehicle) or SB-3CT (final concentrations 0.1-50 μM) in 1% DMSO. At various times, the cells are harvested with trypsin and counted.