Kinase Assays

Kinase activities were assayed in Buffer A or C (unless otherwise stated), at 30℃, at a final ATP concentration of 15 μM. Blank values were subtracted and activities calculated as pmoles of phosphate incorporated during a 10 min. incubation. The activities are usually expressed in % of the maximal activity, i.e. in the absence of inhibitors. Controls were performed with appropriate dilutions of dimethylsulfoxide. In a few cases phosphorylation of the substrate was assessed by autoradiography after SDS-PAGE. GSK-3α/β was purified from porcine brain by affinity chromatography on immobilized axin [S1]. It was assayed, following a 1/100 dilution in 1 mg BSA/ml 10 mM DTT, with 5 μl 40 μM GS-1 peptide, a specific GSK-3 substrate, (YRRAAVPPSPSLSRHSSPHQSpEDEEE, synthesized by the Peptide Synthesis Unit, Institute of Biomolecular Sciences, University of Southampton, U.K.), in buffer A, in the presence of 15 μM [γ-32P] ATP (3,000 Ci/mmol; 1 mCi/ml) in a final volume of 30 μl. After 30 min. incubation at 30℃, 25 μl aliquots of supernatant were spotted onto 2.5 x 3 cm pieces of Whatman P81 phosphocellulose paper, and, 20 sec. later, the filters were washed five times (for at least 5 min. each time) in a solution of 10 ml phosphoric acid/liter of water. The wet filters were counted in the presence of 1 ml ACS (Amersham) scintillation fluid.

Cell lines

Cos-1 cells, SH-SY5Y cells, adult rat mammalian cardiomyocytes

Preparation method

The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reacting condition

5, 10 μM for 24 hr; or 1 μM for 12 hr; 5 μM for 48 hr

Applications

The selective GSK-3 inhibitor BIO inhibited β-catenin phosphorylation on GSK-3-specific sites in Cos-1 cells. Moreover, BIO dramatically decreased level of tyrosine phosphorylation of both GSK-3 isoforms [1]. BIO also increased the proliferation potential of mammalian cardiomyocytes [2].

Animal models

Xenopus laevis embryos model

Dosage form

5, 15 and 50 μM

Applications

BIO is an effective and specific inhibitor of GSK-3 activity in vivo and BIO activated the maternal Wnt signaling pathway in Xenopus laevis embryos [1].

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

GSK-3 inhibitor IX (BIO) is a potent, selective, cell-permeable, ATP-competitive and reversible inhibitor of GSK-3α (glycogen synthase kinase-3α) and GSK-3β (lycogen synthase kinase-3β) (IC50 = 5nM for GSK-3β). [1]

GSK3 (glycogen synthase kinase-3) is a serine/threonine protein kinase which contributes to cell survival, diabetes, insulin resistance and Alzheimer's diseases. It is contributed to β-catenin/Wnt signaling pathway.

BIO facilitated the proliferation in mammalian cardiomyocytes by increasing the proliferation potential of cardiomyocytes. It induced S phase entry and beta-catenin activity in neonatal cardiomyocyte. [3] BIO also activated Wnt signaling and involved in maintaining pluripotency in human and mouse ESCs (embryonic stem cells). [2] In Cos-1 cells treated with 5 μm BIO for 24 hours, phosphorylation of β-catenin was inhibited on GSK-3 specific sites. In cell line deficient for AhR or ARNT, 10 μm BIO treatment for 24 hours showed its effect is through a direct and AhR- independent pathway.[1]

In vivo study showed BIO activated maternal Wnt signaling pathway in Xenopus embryos. It caused a hyper dorso-anteriorization at the expense of trunk and tail in a dose-response manner. It also activated the dorsal genes (siamois and chordin) ectopically. [1]

References:
[1] Meijer L, Skaltsounis AL, Magiatis P, Polychronopoulos P, Knockaert M, Leost M, Ryan XP, Vonica CA, Brivanlou A, Dajani R, Crovace C, Tarricone C, Musacchio A, Roe SM, Pearl L, Greengard P.  GSK-3-selective inhibitors derived from Tyrian purple indirubins.  Chem Biol. 2003 Dec;10(12):1255-66.
[2] Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH.  Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor.  Nat Med. 2004 Jan;10(1):55-63. 
[3] Tseng AS, Engel FB, Keating MT.  The GSK-3 inhibitor BIO promotes proliferation in mammalian cardiomyocytes.  Chem Biol. 2006 Sep;13(9):957-63.