Western blot analysis of extracts from HeLa cells, serum-starved overnight and treated with hIGF-I #8917 (100 ng/ml, 10 min) either with or without Everolimus pretreatment (1 hr) at the indicated concentrations, using Phospho-p70 S6 Kinase (Thr389) Antibody #9205 (upper) or p70 S6 Kinase (49D7) Rabbit mAb #2708 (lower).
Western blot analysis of extracts from HeLa cells, serum-starved overnight and treated with hIGF-I #8917 (100 ng/ml, 10 min) either with or without Everolimus pretreatment (1 hr) at the indicated concentrations, using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb #4858 (upper) or S6 Ribosomal Protein (5G10) Rabbit mAb #2217 (lower).
Chemical structure of everolimus.
Everolimus is supplied as a lyophilized powder. For a 1 mM stock, reconstitute the 1 mg in 1.04 ml DMSO. Working concentrations and length of treatment can vary depending on the desired effect, but it is typically used at 10-100 nM either as a pretreatment for 0.5-1 hr prior to treating with a stimulator or is used alone with varying treatment times lasting up to 24 hr. Soluble in DMSO or ethanol at 100 mg/ml; very poorly soluble in water with maximum solubility ~1-10 µM.
Store lyophilized or in solution at -20ºC, desiccated. Protect from light. 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.
Everolimus, also known as RAD001, is an immunosuppressant analog of rapamycin. Everolimus forms a complex with FKBP12 with an IC50 = ~2 nM in FK506 competitive binding assays (1,2), and this complex then binds to and inhibits mTORC1 (3). Studies have shown that everolimus treatment of cells can lead to the dephosphorylation of mTOR downstream targets (4-7), inhibition of VEGF- and bFRF-stimulated proliferation in HUVE cells (6), and reduction of hypoxia-induced HIF-1 protein levels (7).
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