BIO-acetoxime

Kinases activities are assayed in Buffer A or C, at 30 °C, at a final ATP concentration of 15 μM. Blank values are subtracted and activities calculated as pmoles of phosphate incorporated for a 10 min incubation. The activities are expressed in % of the maximal activity, i.e., in the absence of inhibitors. Controls are performed with appropriate dilutions of DMSO. GSK-3α/β is purified from porcine brain by affinity chromatography on immobilized axin. It is assayed, following a 1/100 dilution in 1 mg BSA/mL 10 mM DTT, with 5 μL 40 μM GS-1 peptide as a substrate, in buffer A, in the presence of 15 μM [γ-³³P] ATP (3000 Ci/mmol; 1 mCi/mL) in a final volume of 30 μL. After 30 min incubation at 30 °C, 25 μL aliquots of supernatant are spotted onto 2.5 × 3 cm pieces of Whatman P81 phosphocellulose paper, and, 20 s later, the filters are washed five times (for at least 5 min each time) in a solution of 10 mL phosphoric acid/liter of water. The wet filters are counted in the presence of 1 mL of ACS scintillation fluid. CDK1/cyclin B is extracted in homogenization buffer from M phase starfish (Marthasterias glacialis) oocytes and purified by affinity chromatography on p9CKShs1-sepharose beads, from which it is eluted by free p9CKShs1. The kinase activity is assayed in buffer C, with 1 mg histone H1 /mL, in the presence of 15 μM [γ-³²P] ATP (3000 Ci/mmol; 1 mCi/mL) in a final volume of 30 μL. After 10 min incubation at 30 °C, 25 μL aliquots of supernatant are spotted onto P81 phosphocellulose papers and treated as described above. CDK5/p25 is reconstituted by mixing equal amounts of recombinant mammalian CDK5 and p25 expressed in E. coli as GST (Glutathione-S-transferase) fusion proteins and purified by affinity chromatography on glutathione-agarose (p25 is a truncated version of p35, the 35 kDa CDK5 activator). Its activity is assayed in buffer C as described for CDK1/cyclin B.0

KCN, KCNR, SY5Y, Kelly, and IMR32 cells

~1 μM

72 h

Cell viability is analyzed by CellTiter 96 AQueous One Solution Cell Proliferation Assay according to manufacturer's instructions, approximately 5,000 cells are plated per well of 96-well tissue culture plates with 100 μL of medium. To assess cell viability, rather than proliferation rate, inhibitor- and control-treated cells are assayed after the same growth time had elapsed. The results represent the mean ± SD of triplicate samples, expressed as a percentage of control.