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Figure 4


Fig. 4. Genistein inhibits EGF-stimulated tyrosine-kinase activity but not ErbB2 internalization. SK-BR-3 cells were serum-starved overnight, treated as described below for the individual panels, fixed and permeabilized. Cells were treated with: (A) GA for 2 hours; (B) 100 ng/ml EGF for 10 minutes; (C) GA for 2 hours, with 100 ng/ml EGF added for the last 10 minutes; and (D) 100 µg/ml genistein for 1 hour, then GA added for another 2 hours and 100 ng/ml EGF added for the last 10 minutes. Deconvolved images from z-stacks are shown. ErbB2 (green, top) and phosphotyrosine (P-Tyr; red, middle) were detected by immunofluorescence. Bottom: merged images. Scale bar: 10 µm. (E) SK-BR-3 cells were pretreated with GA (circles) or GA + 100 µg/ml genistein (squares) for 45 minutes before binding biotinylated anti-ErbB2 antibodies and warming for 0-5 minutes. Internalized antibodies were quantified by CELISA. Values shown are the mean ± s.e.m. of three experiments.





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