Western blot analysis of extracts from MEF cells, untreated (-) or treated with Torin1 (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, or 4E-BP1 Antibody #9452.
Torin 1 is supplied as a lyophilized powder. For a 1 mM stock, reconstitute the 5 mg in 8.23 ml DMSO. First add 1 ml DMSO to the tube containing the chemical, vortex, and dispense into a new, larger tube. Repeat this action two or three more times to transfer any residual material. Add additional DMSO to the new tube to bring the volume up to 8.23 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 1 is a potent and selective ATP-competitive mTOR inhibitor (1,2). Researchers have shown that Torin 1 inhibits mTORC1 and mTORC2 in cell-free kinase assays with IC50 values of 2 nM and 10 nM, respectively (1,2). Torin 1 displays nearly 1000-fold selectivity for mTOR over PI3 Kinase (IC50 ~1.8 μM), 200-fold selectivity over ATM, DNA-PK, and hVps34, and does not significantly inhibit a panel of 353 serine/threonine, tyrosine, and lipid kinases at 10 μM. Torin 1 has also been shown to cause cell cycle arrest via a rapamycin-resistant mechanism independent of mTORC2 (1). Torin 1 treatment of cells leads to the dephosphorylation of mTOR downstream targets including p70 S6 kinase, S6 Ribosomal Protein, and 4E-BP1 (1-4). Activation and nuclear translocation of TFEB (EC50 = ~148 nM) by Torin 1 inhibition of mTORC1 has also been observed (5).
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