Upregulation of EGFR in flotillin depleted MCF7 cells is PI3K-dependent. (A) MCF7 control and flotillin-1 (sh-F1-A/B) or flotillin-2 (sh-F2-A/B) knockdown cells were treated with 1 μM EGFR kinase inhibitor (PD153035) and 1 μM AG9 (control) for 5 minutes prior to stimulation with EGF (100 ng/ml). Western blot with specific antibodies was used to detect pERK1/2, ERK1/2, pMEK1/2, MEK1/2. Loading control: GAPDH. (B) The cells were treated with 20 μM PI3K inhibitor (Ly294002) and DMSO (control) for 24 hours, followed by detection of EGFR, pS473-AKT and AKT. Loading control: GAPDH. (C) Densitometric quantification of EGFR. The signals were normalized to GAPDH. Bars represent the mean ± SD of four independent experiments. Statistical analysis using two-way ANOVA. *, p < 0.05; ***, p < 0.001.