17β-estradiol and EGF induced cell proliferation and raf phosphorylation is mediated through EGFR. (a) SKBR3 and MCF-7 breast cancer cells were pretreated with or without the EGFR antagonist AG1478 (150 nmol/l) 1 hour before treatment with EGF (10 ng/ml) and 17β-oestradiol (10-8 mol/l) alone and in combination for 24 hours. Cell proliferation assays were carried out using MTT thiozolyl blue. Proliferative index of the control group is standardized to 1. The results shown are expressed as mean ± standard error (n = 9). Statistical analysis was performed using Mann-Whitney U test (*P < 0.02 versus without AG1478). (b) Western blot analysis of phospho-Raf and total Raf in SKBR3 and MCF-7 breast cancer cells pretreated with or without the EGFR antagonist AG1478 (150 nmol/l) for 1 hour before 10 minutes of incubation with EGF (10 ng/ml) and 17β-oestradiol (10-8 mol/l) alone and in combination. Results are representative of those obtained in three separate experiments. E2, 17β-oestradiol; EGFR, epidermal growth factor receptor; PI, proliferative index.