GSK 3 Inhibitor IX (6-BIO) is a selective reversible, ATP-competitive inhibitor of GSK-3α/β and CDK1-cyclinB complex. It inhibits (GSK-3α/β)/CDK1/CDK5 activity with IC50 values of 5 nM/320 nM/83 nM, respectively.

CDK5-p35:0.08 μM, TYK2:30 nM, CDK1-CyclinB:0.32 μM, GSK-3:5 nM, CDK2-CyclinA:0.30 μM

BIO抑制小鼠移植瘤模型中黑色素瘤生长.

BIO是pan-JAK抑制剂,作用于TYK2,JAK1,JAK2 和 JAK3,IC50值分别为0.03,1.5,8.0,0.5 μM。BIO是糖原合成酶激酶-3（GSK-3）的特异性抑制剂,GSK-3α/β的IC50为5 nM,比CDK5选择性高16倍以上。BIO与这些激酶的ATP结合口袋相互作用,减少细胞模型中GSK-3特异性位点上的β-连环蛋白磷酸化。BIO促进哺乳动物心肌细胞增殖。BIO选择性抑制STAT3磷酸化,并诱导人类黑色素瘤细胞凋亡。在人类和小鼠胚胎干细胞中,BIO维持未分化表型并维持多能状态特异性转录因子Oct-3/4,Rex-1和Nanog的表达。

Kinase assay: Kinase activities are assayed in Buffer A or C at 30°C, at a final ATP concentration of 15 μM. Blank values are subtracted and activities calculated as pmoles of phosphate incorporated during a 10 min incubation. Controls are performed with appropriate dilutions of dimethylsulfoxide. In a few cases phosphorylation of the substrate is assessed by autoradiography after SDS-PAGE. GSK-3α/β is purified from porcine brain by affinity chromatography on immobilized axin. It is 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), 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°C, 25 μl aliquots of supernatant are spotted onto 2.5 × 3 cm pieces of Whatman P81 phosphocellulose paper, and 20 seconds later, the filters are washed five times (for at least 5 min each time) in a solution of 10 ml phosphoric acid/liter of water. The wet filters are counted in the presence of 1 ml ACS scintillation fluid.

COS1, Hepa (wild-type, CEM/LM AhR deficient and ELB1 ARNT deficient), or SH-SY5Y cells are grown in 6 cm culture dishes in Dulbecco's Modified Medium (DMEM) containing 10% fetal bovine serum. For treatment, IO (5 μM), BIO (5 or 10 μM), MeBIO (5 or 50 μM), LiCl (20 or 40 mM), or mock solution (DMSO, 0.5% final concentration) is added to medium when cell density reaches ～70% confluence. After 12 (SH-SY5Y) or 24 hours, the cells, while still in plate, are lysed with lysis buffer (1% SDS, 1 mM sodium orthovanadate, 10 mM Tris [pH 7.4]). The lysate is passed several times through a 26 g needle, centrifuged at 10,000 × g for 5 min, and adjusted to equal protein concentration. About 8 μg of each sample is loaded for immunoblotting. Enhanced chemiluminescence is used for detection. The following primary antibodies are used: mouse anti-β-catenin CT (Upstate Biotechnolgies, Clone 7D8, recognizes total β-catenin), mouse anti-phospho-β-catenin (Upstate Biotechnologies, Clone 8E7, recognizes dephosphorylated β-catenin), mouse anti-GSK-3 β, mouse anti-GSK-3 phosphoTyr216, rabbit anti-AhR (Aryl hydrocarbon receptor), and rabbit anti-actin.(Only for Reference)