Figure 4
Western Blot analysis of PI3K/AKT and RAS-RAF-MAPK signalling. The effect of EGF and SDF-1α on induction of PI3K/AKT and RAS-RAF-MAPK signalling was analysed by Western Blot. Cells were stimulated for 5 minutes with 100 ng/ml EGF and 1 μg/ml SDF-1α. In case of inhibitor treatment cells were preincubated for 30 minutes with 500 nM Ly294002 and 500 nM PD98059. The downstream effect of EGF and SDF-1α stimulation on the AKT and MAP-kinase pathways was determined by phosphorylation of AKT and MAPKp42/44. To investigate for the putative RAF-AKT crosstalk RAF-1 expression and RAF-1 phosphorylation at position S259 was determined. The arrow marks pRAF-1 S259. elf4E served as loading control. Shown are representative Western-Blot data of three independent experiments. A) M13SV1-EGFP-Neo breast epithelial cells, B) M13MDA435-1 hybrid cells, C) M13MDA435-3 hybrid cells, D) MDA-MB-435-Hyg breast cancer cells. Bands were densitometric analysed by using the ImageJ software http://rsbweb.nih.gov/ij/. Relative intensities of protein and phosphoprotein expression were calculated in relation to elf4E and controls, which were set to 100%.