Inhibition of IGF-IR/InsR or PI3K abrogates AZD5363-induced AKT membrane localization and phosphorylation. A) MCF-7/LTED cells were transfected with an AKT PH-GFP plasmid. On day four, cells were treated with 100 ng/ml IGF-I in serum-free medium for 15 minutes, or pre-incubated with 10% DCC-FBS ± 1 µM AEW541 or 1 µM BKM120 for 30 minutes followed by treatment with 2 µM AZD5363 for four hours. Cells were viewed in a LSM 510Meta confocal microscope at 40x magnification. B) LTED lines were treated with 10% DCC-FBS ± 1 µM AEW541 or BKM120 for 1 hour, followed by the addition of 2 µM AZD5363 for 24 hours. Protein lysates were analyzed by immunoblot using the indicated antibodies. DCC-FBS, dextran/charcoal-treated fetal bovine serum; IGF-I, insulin-like growth factor-I; LTED, long-term estrogen deprivation.