BrCa cell cultures were treated with AAE for 24 h, cells were harvested and cell extracts were analyzed by immunoblotting. The levels of total Akt and mTOR, phospho-Akt, phospho-mTOR, total S6 ribosomal protein and 4E-BP1, phospho-S6 and phospho-4E-BP1, total ULK1 and phospho-ULK1, as well as ATG13 were detected by immunoblotting. Positive control used for panel A is AKT inhibitor, SH-5 at 20 μM; for panel B is mTOR inhibitor rapamycin at 25 nM. Levels of β-actin was used as a loading control. Since protein lysates from same experiment were analyzed for detecting signaling proteins, a single β-actin blot is shown at the bottom. All blots were repeated using new samples twice, with comparable results.