P erbb3

Mammary glands and cells were homogenized in ice-cold 50 mM Tris (pH 7.4), 100 mM NaF, 120 mM NaCl, 0.5% Nonidet P-40, 100 μM Na₃VO₄, and 1× protease inhibitor mixture (Roche), sonicated for 10 s on ice, and cleared by centrifugation at 4 °C, 13,000 × g for 20 min. Protein concentration was determined using the bicinchoninic acid assay (Pierce). Proteins were separated by SDS/PAGE and transferred to nitrocellulose membranes. Membranes were blocked in 3% gelatin in TBS-T (Tris-buffered saline, 0.1% Tween-20) for 1 h, incubated in primary antibody in 3% gelatin overnight at 4 °C, washed with TBS-T, incubated in HRP-conjugated anti-rabbit (sc-2375, 1:10,000; Santa Cruz Biotechnology) or anti-mouse IgG (ab97240, 1:5000; Abcam), washed with TBS-T, and then developed using ECL substrate (Pierce). The following primary antibodies were used: P-ErbB3 (sc-135654, 1:500; Santa Cruz Biotechnology), P-MAPK/Akt/S6/Rab11 cocktail (1:1000; Cell Signaling Technologies), total Akt (C67E7, 1:1000; Cell Signaling Technologies), actin (1:2000; Cell Signaling), STAT5A and P-STAT5A/B (93585 at 1:500 and C11C5 at 1:500, respectively; Cell Signaling Technologies), and cleaved caspase 3 (D175, 1:500; Cell Signaling Technologies).

NVP-AEW541 and NVP-BEZ235 were gifts from Novartis (Basel, Switzerland). Lapatinib, KU0063794 and LY294002 were from Cayman Chemical (Michigan, USA). Cisplatin was purchased from Sigma-Aldrich (Steinheim, Germany). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Serva (Heidelberg, Germany). Propidium iodide was from PromoCell (Heidelberg, Germany). Roswell Park Memorial Institute (RPMI) media 1640, Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin [10,000 U/ml; 10 mg/ml] and trypsin-EDTA (0.05% trypsin, 0.02% EDTA in PBS) were purchased from PAN Biotech (Aidenbach, Germany). Primary antibodies were purchased from R&D Systems (Wiesbaden, Germany) (pIGF1R, IGF1R, p-EGFR, EGFR, p-ErbB2, ErbB2, p-ErbB3, ErbB3) or Santa Cruz Biotechnology (Heidelberg, Germany) (p-Akt, Akt, β-Actin, PARP). HRP-conjugated secondary antibodies were from R&D Systems. All other reagents and chemicals were from VWR BDH PROLABO (Darmstadt, Germany).