EGF stimulation of MCF7 cells depleted of flotillin-1 or flotillin-2. (A) Western blot with specific antibodies for pY1173-EGFR, EGFR, pS473-AKT, AKT, pS338-Raf1, Raf1, pMEK1/2, MEK1/2, pERK1/2, ERK1/2, flotillin-1, flotillin-2 and GAPDH in MCF7 cells depleted of flotillin-1 (sh-F1-B) or flotillin-2 (sh-F2-A) after EGF-stimulation (100 ng/ml) for 10 and 30 minutes. (B-G) Densitometric quantification of EGFR (B), pY1173-EGFR (C, D), pMEK1/2 (E), pERK1/2 (F) and pS338-Raf1 (G). The signals for total protein were normalized to GAPDH, phosphorylated proteins to the corresponding total protein. Bars represent the mean ± SD of four independent experiments. Statistical analysis using two-way ANOVA. *, p < 0.05; **, p < 0.01.