SB 415286

In Vitro

Aliquot the stock solution and store it at -80°C (up to 2 years) or -20°C (up to 1 year); avoid repeated freeze-thaw cycles.

In Vivo

Choose the formulation that suits the animal model and route of administration; percentages are volume ratios of the final working solution. Start from a clear DMSO stock (see In Vitro above), add the co-solvents one at a time in the order listed, mixing after each addition, and prepare the working solution fresh on the day of dosing. If precipitation or phase separation occurs, gentle warming or sonication can help.

Data provided by the manufacturer.

In Vitro

SB 415286 (SB-415286) blocks human GSK-3α, with an IC₅₀ of 77.5 nM and a K_i of 30.75 nM. SB-415286 promotes glycogen synthesis in the Chang human liver cell line, with an EC₅₀ of 2.9 μM. SB-415286 also raises glycogen synthase activity in Chang human liver cells. SB-415286 drives transcription of a β-catenin-LEF/TCF regulated reporter gene in HEK293 cells^[1]. In B65 cells, SB 415286 (SB-415286, 5-44 μM) reduces cell loss caused by 1 mM H₂O₂. SB-415286 (5-44 μM) produces a significant dose-dependent drop in DCF fluorescence intensity and reduces B65 ROS production induced by 1 mM H₂O₂. SB-415286 (5-44 μM) likewise reduces ROS production in CGN induced by 1 mM H₂O₂^[2]. SB-415286 (50 μM) substantially suppresses immunoprecipitated GSK3 activity, by 97%^[3].

Data provided by the manufacturer. Numbered citations refer to the Literature Cited list in the product description.

Kinase Assay^[1]

Measure GSK-3 kinase activity, with or without SB-216763 or SB-415286, in a reaction mixture made up of the following final concentrations: human GSK-3α or rabbit GSK3α at 1 nM; MOPS at 50 mM, pH 7.0; with EDTA at 0.2 mM and Mg-acetate at 10 mM; β-mercaptoethanol at 7.5 mM; together with glycerol at 5% (w/v) and Tween-20 at 0.01% (w/v); DMSO at 10% (v/v); and finally GS-2 peptide substrate at 28 μM (the sequence of the GS-2 peptide matches a glycogen synthase region that GSK-3 phosphorylates). Start the assay by adding 0.34 μCi [³³P]γ-ATP (IC₅₀ determinations) or 2.7 μCi [³³P]γ-ATP (K_i determinations). Set total ATP at 10 μM (IC₅₀ determinations) or from 0 to 45 μM (K_i determinations). Incubate 30 min at room temperature, then stop the assay with one third assay volume of 2.5% (v/v) H₃PO₄ containing 21 mM ATP. Spot samples onto P30 phosphocellulose mats and wash the mats six times in 0.5% (v/v) H₃PO₄. Seal the filter mats in sample bags containing Wallac betaplate scintillation fluid. Count the mats in a Wallac microbeta scintillation counter to determine ³³P incorporation into the substrate peptide^[1].

Cell Assay^[2]

Use B65 cells after 24 h of in vitro culture and CGN after 7-8 days in vitro. Dissolve lithium in culture media and SB-415286 in DMSO, and add them to the neuronal preparation at the precise concentrations 1 h before adding H₂O₂ (50 μM to 1 mM). Assess the loss in cell viability with the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium] method. Add MTT to the cells at a final concentration of 250 μM and incubate for 1 h so that MTT reduction yields a dark blue formazan product. Remove the media, then dissolve the cells in dimethylsulfoxide. Measure formazan production by the absorbency change at 595 nm using a microplate reader. Express viability results as percentages, taking the absorbency of non-treated cells as 100%^[2].

Data provided by the manufacturer. Numbered citations refer to the Literature Cited list in the product description.