Figure 1

AnxA6 is required for the localization of activated EGFR on the surface of breast cancer cells. A) BT-549, HCC1806 and MDA-MB-468 cells were grown to 70% confluency, followed by serum starvation for 24 h. Cells were then treated with EGF for 0–90 min and harvested by scrapping in ice-cold PBS. Equal amounts of whole cell lysates were separated in 4-12% polyacrylamide gels under reducing conditions and analyzed by Western blotting with the indicated antibodies. B) Densitometric analysis of EGFR expression. Bars represent EGFR expression relative to BT-549 cells from at least three independent experiments. C) Densitometric analysis of EGF-induced EGFR activation. Points represent activated EGFR (p-Y1068) relative to untreated control cells from at least three independent experiments. D) AnxA6 expression promotes a sustained cell surface localization of activated EGFR in breast cancer cells. BT-549, HCC1806 and MDA-MB-468 cells were grown on glass cover slips in complete DMEM/F12 medium, then serum-starved overnight. The cells were subsequently washed twice in Hanks’ balanced salt solution (HBSS) and then treated with or without EGF for 5 min. The cells were then fixed for 20 min in 3% paraformaldehyde in PBS. Activated EGFR was detected by immunofluorescence staining with antibodies to phospho-EGFR (Y1068) and counterstained with DAPI. Bars = 10 μm.