Analysis of phosphatidylinositol-3-kinase pathway signaling in breast cancer cells. Cell lines were grown to subconfluency and placed in medium containing low (0.5%) fetal bovine serum overnight prior to preparation of cell lysates. Equal amounts (25 μg) of extracted protein from each cell line were then immunoblotted using antibodies against the indicated proteins. P-Tyr1248 HER2 (p-HER2); p-Ser473 Akt (p-Akt); p-Thr389 S6 protein kinase 1 (p-S6K1); p-Ser235/236 S6 (p-S6); p-Thr202/Tyr204 ERK (p-ERK). ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; PTEN, phosphatase and tensin homolog; mTOR, mammalian target of rapamycin.