Fig. 2

EGFR activity promotes constitutive Gab1 and MAPK phosphorylation in MDA-MB-468 cells. A MDA-MB-468 cells were seeded and cultivated in phenol red-free RPMI 1640 for 24 h. On the following day, cells were treated with DMSO or Gefitinib (3 µM) for 30 min. Subsequently, cells were lysed and proteins were separated by SDS-PAGE. After Western blotting, membranes were stained with specific antibodies against (p)Y Gab1, Gab1, (p)T/Y ERK1/2, ERK1/2, (p)S Akt, Akt, (p)Y STAT3, STAT3 and tubulin. A representative result of n = 3 independent experiments is shown. The results from (A) were quantified. The diagrams show the ratios of B (p)Y Gab1 to Gab1, C (p)T/Y ERK1/2 to ERK1/2, D (p)S Akt to Akt, and E (p)Y STAT3 to STAT3. Gab1, ERK1/2, Akt and STAT3 phosphorylation in DMSO-treated cells was normalised to 100% in each independent repetition of the experiment. Data are given as mean of three independent experiments ± SD. Student’s t-test: n.s. = non-significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001. F MDA-MB-468 cells were seeded on poly-L-lysine-coated glass cover slips and cultivated in phenol red-free RPMI 1640. After 24 h, cells were transfected with an expression vector for murine Gab1-GFP. On the following day, cells were placed into the incubation chamber of a laser scanning microscope and left for 30 min. Cells were treated with Gefitinib (3 µM) or DMSO for 30 min. Imaging was performed after treatment with Gefitinib or DMSO. Representative results of n = 3 independent experiments are shown