Western blot analysis of extracts from NIH/3T3 cells, untreated or treated with serum in the presence of the indicated concentrations of Rapamycin, using Phospho-p70 S6 Kinase (Thr389) Antibody #9205 (upper), total p70 S6 kinase antibody #9202 (middle) or Phospho-p44/42 MAPK (Thr202/Tyr204) Antibody #9101 (lower).
Rapamycin is supplied as a lyophilized powder. For a 100 µM stock, resuspend 10 nmol (9.1 µg) in 100 µl ethanol or DMSO. For experiments with cultured cells, CST recommends pretreating with 10 nM of this inhibitor for one hour prior to stimulation.
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.
Rapamycin is a bacterial macrolide with antifungal and immunosuppressant activities (1). Rapamycin forms a complex with the immunophilin FKBP12 which then inhibits the activity of FRAP/ mTOR (TOR in yeast) (2,3). Rapamycin treatment of cells leads to the dephosphorylation and inactivation of p70 S6 kinase. Rapamycin also leads to the dephosphorylation of 4E-BP1/PHAS1, thereby promoting its binding to and inactivation of eIF4E (4,5). This activity has been shown to be the basis for Rapamycin's ability to block protein synthesis and to arrest cell cycle progression in the G1-phase (6,7). However, it has been suggested that Rapamycin's inhibition of the G1/S transition may be the consequence of its effect on cyclin D1 mRNA and protein stability (8).
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