Western blot analysis of extracts from MCF7 cells, serum-starved overnight and untreated (-) or treated with hIGF-I #8917 (100 ng/ml, 10 min; +), either with or without Torin 2 pretreatment (1 hr) at the indicated concentrations, using Phospho-p70 S6 Kinase (Thr389) (108D2) Rabbit mAb #9234, p70 S6 Kinase (49D7) Rabbit mAb #2708, Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb #4858, S6 Ribosomal Protein (5G10) Rabbit mAb #2217, Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb #2855, and 4E-BP1 Antibody #9452.Learn more about how we get our images
Torin 2 is supplied as a lyophilized powder. For a 5 mM stock, reconstitute the 5 mg in 2.31 ml DMSO. First add 1 ml DMSO to the tube containing the chemical, vortex, and dispense into a new, larger tube. Repeat this action to transfer any residual material. Add additional DMSO to the new tube to bring the volume up to 2.31 ml. Heating to 37ºC and/or additional vortexing may be required.
Working concentrations and length of treatment can vary depending on the desired effect, but it is typically used at 10-1,000 nM for 1-24 hr.
Store lyophilized or in solution at -20ºC, desiccated. In lyophilized form, the chemical is stable for 24 months. Once in solution, use within 3 months to prevent loss of potency. Aliquot to avoid multiple freeze/thaw cycles.
Torin 2 is a potent and selective ATP-competitive mTOR inhibitor with superior pharmacokinetics over the Torin 1 predecessor (1,2). A series of in vitro kinase activity assays indicate that Torin 2 inhibits mTORC1 with an IC50 of 2.1 nM. Cellular activity assays demonstrate that Torin 2 inhibits cellular mTOR activity with an EC50 of 0.25 nM. These assays also indicate that Torin 2 has an 800-fold selectivity over P13K (EC50 of 200 nM) and a over 800-fold selectivity over 400 other protein kinases (2). Unlike Torin 1, Torin 2 inhibits the phosphatidylinositol-3 kinase–like kinase (PIKK) family members ATM (EC50 = 25 nM), ATR (EC50 = 35 nM), and DNA-PK (IC50 = 118 nM) (2). Investigators demonstrate that Torin 2 treatment of cells attenuates phosphorylation of mTOR downstream targets, inhibits cell proliferation of several cancer cell types, and induces apoptosis and autophagy (2,3). Indirect activation and nuclear translocation of TFEB (EC50 = ~1.6 nM) through Torin 2 inhibition of mTORC1 has also been observed (4).
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