The human mammary carcinoma cell lines BT474, SKBR3, HCC1419, MDA-MB453, UACC893, and MDA-MB361 were obtained from the American Type Culture Collection (www.atcc.org) and cultured in RPMI 1640 medium from Life Technologies with 1% penicillin streptomycin from Sigma-Aldrich and 10% fetal calf serum from PAA Laboratories. Cells were seeded at a density of 10,000 cells/cm2 24 hours before treatment. Protease inhibitors Pefabloc from Merck, leupeptin and pepstatin from Serva, and marimastat from Calbiochem and the phosphatase inhibitors sodium orthovanadate, sodium metavanadate, sodium molybdate, sodium β-glycerol phosphate, and sodium fluoride from Sigma-Aldrich were used for Western blot analysis. Antibodies against ErbB3 (1:1000; no. 12708), p-ErbB3 (Y1289) (1:500; no. 4791), Erk1/2 (1:1000; no. 9102), p-Erk1/2 (T202/Y204) (1:1000; no. 4370), pan-AKT (1:2000; no. 4691), p-AKT (S473) (1:2000; no. 4060), PARP (1:1000; no. 9542), FAK (1:500; no. 71433), and p-FAK (Y397) (1:500; no. 8556) were purchased from Cell Signaling Technology. Anti-ErbB2 (3B5) (1:5000; OP15) and p-ErbB2 (Y1248) (1:10,000; 06-229) were from Calbiochem. An antibody against Glyceraldehyde-3-phosphate dehydrogenase (1:2000; sc-365062) was purchased from Santa Cruz Biotechnology. Secondary anti-mouse immunoglobulin G (IgG) IR-Dye800 conjugate from Rockland (610-732-124) and anti-rabbit IgG Alexa Fluor 680 (A-21109) from Invitrogen was purchased (1:10,000) for infrared Western blot detection on an Odyssey system from LI-COR. ARRY380, MK2206, and PF5622271 were obtained from Selleckchem. DARPin expression and purification have been described previously (38). TZB (Herceptin) was obtained from Kantonsapotheke Zürich.

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