IM-12

IM-12 is an inhibitor of GSK-3β, with an IC50 of 53 nM, and also enhances Wnt signalling.

Signaling Pathways >> PI3K/Akt/mTOR >> GSK-3

Signaling Pathways >> Stem Cell/Wnt >> GSK-3

Research Areas >> Cancer

GSK-3β:53 nM (IC50)

IM-12 inhibits GSK-3β in ReNcell VM cells, with I50 of 3.8 μM. IM-12 (3 μM) enhances the β-catenin amount, with no further effect at lower or higher concentration. IM-12 (3 μM) also attenuates the proliferation of ReNCell VM cells. IM-12 increases TCF-activity of ReNcell VM[1].

Cells are lysed in RIPA buffer, supplemented with protease and phosphatase inhibitors and centrifuged for 5 min at 15,000 rpm. Immunoprecipitation of GSK-3β is performed with a specific mouse monoclonal anti GSK-3β [G8] antibody with 5 μg/sample for 2 h at 4°C. The bound protein is precipitated with Protein A/G-Plus agarose-beads (10 μL beads per sample). GSK-3β kinase activity is measured in a reaction mixture containing final concentrations of: 4 mM MOPS pH 7.2; 0.4 mM EDTA; 1 mM EGTA; 2.5 mM β-glycerophosphate; 4 mM MgCl2; 40 μM BSA; 0.05 mM DTT. 10 μg/sample pGS-2 peptide substrate is used[1].

To measure viable cells, 50-100 μL of cell suspension is analyzed using CASY technology with the appropriate program. ReNcell VM cells are seeded at a defined cell number and proliferated for 24 h. Then the medium is changed to proliferation medium with added substances at indicated concentrations. The cell number is determined every 24 h. Cells are exposed to the added drugs during the whole experiment, whereas the media is changed every 24 h[1].

[1]. Schmole AC, et al. Novel indolylmaleimide acts as GSK-3beta inhibitor in human neural progenitor cells. Bioorg Med Chem. 2010 Sep 15;18(18):6785-95.

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