Fig. 4

MAPK and PI3K activity promote constitutive Gab1 phosphorylation in MDA-MB-468 cells. A MDA-MB-468 cells were cultivated in phenol red-free RPMI 1640 for 24 h. On the following day, cells were treated with DMSO, U0126 (10 µM) and/or LY294002 (40 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. One-way ANOVA: n.s. = non-significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001