Phospho erαser118

Taselisib, also called GDC-0032, was generated at Genentech, Inc. (South San Francisco, CA). Letrozole was obtained from US Biological. Antibodies used include phospho-AKT^Ser473, AKT, phospho-PRAS40^Thr246, phospho-S6^Ser235/236, phospho-S6^Ser240/242, S6, phospho-ERK^{Thr202/Tyr204}, ERK, phospho-ERα^Ser118, phospho-ERα^Ser167, cleaved PARP, p110α, phospho-p70S6K^Thr389, PR, cyclin E, phospho-mTOR^Ser2448, IGF1R, BRCA1, c-Myc, CAV1, HER2 and cyclin D1 obtained from Cell Signaling (Danvers, MA). Antibodies for ERα and ERβ were obtained from Santa Cruz biotechnology (Santa Cruz, CA) and a βActin antibody was obtained from Sigma (St. Louis, MO).

Breast cancer cell lines were lysed in radioimmunoassay buffer (RIPA) and equal amounts of protein were subjected to SDS-PAGE and Western blot analysis in triplicates as described previously [32 (link)]. The Bio-Rad DC-Protein assay kit (Bio-Rad, Hercules, CA) was used to determine protein concentrations. Blots were incubated with antibodies against DLC1 (Thermo fisher Scientific, Waltham, MA), ECT2 (EMD Millipore, Burlington, MA), phospho-ECT2 [kind gift of Dr. Alan Fields [33 (link)]]. Antibodies against TFF1/pS2, cyclin D1, c-Myc, estrogen receptor α (ERα) and phospho-ERα (Ser118) were purchased from Cell Signaling Technology (Danvers, MA). Phospho-specific ECT2 antibody production and validation were described as previously [33 (link)]. Briefly, immunogenic phosphopeptides were: Ac-321CYLYEKANpTPELKKSV335-amide and Ac-321YLYEKANpTPELKKSVC335-amide respectively. Antibodies against GAPDH (GeneTex, Inc., Irvine, CA) and β-actin Sigma (St. Louis, MO) were used as loading controls. Protein bands were visualized by SuperSignal West Pico PLUS Chemiluminescent Substrate kit (Amersham, Piscataway, NJ) and Amersham Imager 600 GE Healthcare Life Sciences (GE Healthcare Bio-Sciences, Pittsburgh, PA). The data are representative of three individual sets.