Figure S1 - HER2 Oncogenic Function Escapes EGFR Tyrosine Kinase Inhibitors via Activation of Alternative HER Receptors in Breast Cancer Cells

Inhibition of EGFR with TKI AG 1478 does not abolish HER2 phosphorylation. A, A431, MCF-7, MDAMB-453 and SKBR3 cells were grown to near confluency before lysis for western blot analysis. The membrane was probed with either anti-HER2 or anti-EGFR antibody. B, A431 cells pre-treated with increasing doses of AG 1478 for two hours before being stimulated with 100 ng/ml EGF for 10 minutes. The cells were assessed for HER2 phosphorylation by FRET. C, A431 cells were pre-treated by increasing doses of AG 1478 as illustrated before 100 ng/ml EGF stimulation and western blot analysis. The phosphorylation of PKB on Ser473 and Erk1/Erk2 (p44/42 MAP Kinase) on Thr202/Tyr204 was determined using phosphospecific antibodies. The total endogenous levels of Erk1/Erk2 were assessed by western blot using anti-ERK antibodies. D, Upper panels, A431 cells and two other breast cancer cell lines MDAMB-453 and SKBR3 cells were assessed for HER2 phosphorylation after pre-treatment of the cells with 3 µM AG 1478 for two hours. Lower panels, A431, MDAMB-453 and SKBR3 cells were lysed for western blot analysis after treatment with either 3 µM AG 1478 or vehicle for two hours. The phosphorylation of HER2, phosphoPKB Ser473 and Erk1/Erk2 was determined using phosphospecific antibodies
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