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FIG. 7. Inhibition of EGFR opposes Tam-induced ERK1/2 phosphorylation in both MCF-7 and T47D cells. MCF-7 cells were precultured for 2 d in RPMI 1640 medium containing dcFBS (5%) in the absence of E2, after which either vehicle or 10 µM AG1478 (A) or 10 µM BIBX 1382 (B) were added for 1 h. The cells were then incubated in serum-free medium containing DMSO (0.2%), 10 nM E2, 5 µM Tam, or a combination of 5 µM Tam and 10 nM E2 for 30 min. Whole-cell extracts were prepared and 30-µl aliquots of whole cell lysate per lane were analyzed by Western blotting, using antibodies against P-ERK1/2 and ERK1/2. Quantification of band intensities was done by MCID Image Analyzer and fold stimulation (compared with vehicle) was calculated and the graph represents values of two or three independent experiments. Alternatively, T47D cells were pretreated for 1 h with 10 µM BIBX 1382 after which cells were treated and ERK-phosphorylation assessed as previously (B). *, P < 0.05. For statistical analyses AG1478 and BIBX 1282-pretreated samples were compared with corresponding vehicle-pretreated samples.
