GSK-3 kinase activity is measured, in the presence or absence of SB-216763 or SB-415286, in a reaction mixture containing final concentrations of: 1 nM human GSK-3α or rabbit GSK3α; 50 mM MOPS pH 7.0; 0.2 mM EDTA; 10 mM Mg-acetate; 7.5 mM β-mercaptoethanol; 5% (w/v) glycerol; 0.01% (w/v) Tween-20; 10% (v/v) DMSO; 28 μM GS-2 peptide substrate. The GS-2 peptide sequence corresponds to a region of glycogen synthase that is phosphorylated by GSK-3. The assay is initiated by the addition of 0.34 μCi [³³P]γ-ATP (IC₅₀ determinations) or 2.7 μCi [³³P]γ-ATP (K_i determinations). The total ATP concentration is 10 μM (IC₅₀ determinations) or ranges from 0 to 45 μM (K_i determinations). Following 30 min incubation at room temperature the assay is stopped by the addition of one third assay volume of 2.5% (v/v) H₃PO₄ containing 21 mM ATP. Samples are spotted onto P30 phosphocellulose mats and these are washed six times in 0.5% (v/v) H₃PO₄. The filter mats are sealed into sample bags containing Wallac betaplate scintillation fluid. ³³P incorporation into the substrate peptide is determined by counting the mats in a Wallac microbeta scintillation counter^[1].

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

B65 cells are used after 24 h of in vitro culture. CGN are used after 7-8 days in vitro. Lithium and SB-415286 are dissolved in culture media and DMSO, respectively, and added to the neuronal preparation at the precise concentrations, 1 h before addition H₂O₂ (50 μM to 1 mM). To assess the loss in cell viability, we use the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium] method. MTT is added to the cells at a final concentration of 250 μM and incubated for 1 h, allowing the reduction in MTT to produce a dark blue formazan product. Media are then removed, and cells are dissolved in dimethylsulfoxide. Formazan production is measured by the absorbency change at 595 nm using a microplate reader. Viability results are expressed as percentages. The absorbency measured from non-treated cells is taken to be 100%^[2].

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

• [1]. Coghlan MP, et al. Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription. Chem Biol. 2000 Oct;7(10):793-803.  [Content Brief]

  [2]. Pizarro JG, et al. Neuroprotective effects of SB-415286 on hydrogen peroxide-induced cell death in B65 rat neuroblastoma cells and neurons. Int J Dev Neurosci. 2008 May-Jun;26(3-4):269-76.  [Content Brief]

  [3]. MacAulay K, et al. Use of lithium and SB-415286 to explore the role of glycogen synthase kinase-3 in the regulation of glucose transport and glycogen synthase. Eur J Biochem. 2003 Sep;270(18):3829-38.  [Content Brief]