CHIR-98014 

Polypropylene 96-well plates are filled with 300 μL/well buffer (50 mM tris HCl, 10 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 25 mM β-glycerophosphate, 1 mM NaF, 0.01% BSA, pH 7.5) containing kinase, peptide substrate, and any activators. Information on the kinase concentration, peptide substrate, and activator for these assays is as follows: GSK-3α (27 nM, and 0.5 μM biotin-CREB peptide); GSK-3β (29 nM, and 0.5 μM biotin-CREB peptide); cdc2 (0.8 nM, and 0.5 μM biotin histone H1 peptide); erk2 (400 units/mL, and myelin basic protein-coated Flash Plate); PKC-α (1.6 nM, 0.5 μM biotin-histone H1 peptide, and 0.1 mg/mL phosphatidylserine + 0.01 mg/mL diglycerides); PKC-ζ (0.1 nM, 0.5 μM biotin-PKC-86 peptide, and 50 μg/mL phosphatidylserine + 5 μg/mL diacylglycerol); akt1 (5.55 nM, and 0.5 μM biotin phospho-AKT peptide); p70 S6 kinase (1.5 nM, and 0.5 μM biotin-GGGKRRRLASLRA); p90 RSK2 (0.049 units/mL, and 0.5 μM biotin-GGGKRRRLASLRA); c-src (4.1 units/mL, and 0.5 μM biotin-KVEKIGEGTYGVVYK); Tie2 (1 μg/mL, and 200 nM biotin-GGGGAPEDLYKDFLT); flt1 (1.8 nM, and 0.25 μM KDRY1175 [B91616] biotin-GGGGQDGKDYIVLPI-NH2); KDR (0.95 nM, and 0.25 μM KDRY1175 [B91616] biotin-GGGGQDGKDYIVLPI-NH2); bFGF receptor tyrosine kinase (RTK; 2 nM, and 0.25 μM KDRY1175 [B91616] biotin-GGGGQDGKDYIVLPI-NH2); IGF1 RTK (1.91 nM, and 1 μM biotin-GGGGKKKSPGEYVNIEFG-amide); insulin RTK (using DG44 IR cells); AMP kinase (470 units/mL, 50 μM SAMS peptide, and 300 μM AMP); pdk1 (0.25 nM, 2.9 nM unactivated Akt, and 20 μM each of DOPC and DOPS + 2 μM PIP3); CHK1 (1.4 nM, and 0.5 μM biotin-cdc25 peptide); CK1-ε (3 nM, and 0.2 μM biotin-peptide); DNA PK (see 31); and phosphatidylinositol (PI) 3-kinase (5 nM, and 2 μg/mL PI). Test compounds or controls are added in 3.5 μL of DMSO, followed by 50 μL of ATP stock to yield a final concentration of 1 μM ATP in all cell-free assays. After incubation, triplicate 100-μL aliquots are transferred to Combiplate eight plates containing 100 μL/well 50 μM ATP and 20 mM EDTA. After 1 h, the wells are rinsed five times with PBS, filled with 200 μL of scintillation fluid, sealed, left 30 min, and counted in a scintillation counter. All steps are performed at room temperature. Inhibition is calculated as 100% × (inhibited − no enzyme control)/(DMSO control − no enzyme control)^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

The viability of the mouse ES cells is determined after exposure to different concentrations of GSK3 inhibitors for three days using the MTT assay. The decrease of MTT activity is a reliable metabolism-based test for quantifying cell viability; this decrease correlates with the loss of cell viability. 2,000 cells are seeded overnight on gelatine-coated 96-well plates in LIF-containing ES cell medium. On the next day the medium is changed to medium devoid of LIF and with reduced serum and supplemented with 0.1-1 μM BIO, or 1-10 μM SB-216763, CHIR-99021 or CHIR-98014. Basal medium without GSK3 inhibitors or DMSO is used as control. All tested conditions are analyzed in triplicates^[2].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Blood is obtained by shallow tail snipping at lidocaine-anesthetized tips. Blood glucose is measured directly or heparinized plasma is collected for measurement of glucose or insulin. Animals are prebled and randomized to vehicle control or GSK-3 inhibitor treatment groups. For glucose tolerance tests (GTTs), animals are fasted throughout the procedure with food removal early in the morning, 3 h before first prebleed (db/db mice), or the previous night, 16 h before the bleed (ZDF rats). When the time course of plasma glucose and insulin changes in fasting ZDF rats is measured, food is removed ∼16 h before test agent administration. The glucose challenges in the GTT are 1.35 g/kg i.p. (ipGTT) or 2 g/kg via oral gavage (oGTT). Test inhibitors are formulated as solutions in 20 mM citrate-buffered 15% Captisol or as fine suspensions in 0.5% carboxymethylcellulose^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Ring DB, et al. Selective glycogen synthase kinase 3 inhibitors potentiate insulin activation of glucose transport and utilization in vitro and in vivo. Diabetes. 2003 Mar;52(3):588-95.  [Content Brief]

  [2]. Naujok O, et al. Cytotoxicity and activation of the Wnt/beta-catenin pathway in mouse embryonic stem cells treated with four GSK3 inhibitors. BMC Res Notes. 2014 Apr 29;7:273.  [Content Brief]

  [3]. Zajkowski T, et al. Stabilization of microtubular cytoskeleton protects neurons from toxicity of N-terminal fragment of cytosolic prion protein. Biochim Biophys Acta. 2015 Oct;1853(10 Pt A):2228-39.  [Content Brief]