EGFR Activation under Oxidative Stress Figure 6. Oxidative stress induces a novel active conformation of EGFR. A. A549 cells were treated (or not) with 100 ng/ml EGF for 15 min. or with 1 U/ml GO for 30 min; Tyr-phosphorylation (p-Y20) and total EGFR levels were assessed by IB, as indicated. B. EGFR was IPed from 300 mg of the total cell lysates using either a528 or a4-2 Ab and then IBed with aEGFR Ab (2232). Samples IBs and the IgG heavy chains (stained with amouse Ab) of the Abs used in the IPs are shown. The histogram in C represents the averaged ratio between total EGFR (IPed with a528 Ab) and the EGFR IPed with the a4-2 Ab (which has affinity for the ‘‘classical’’ EGF-induced active receptor conformation) of three independent experiments, quantified by densitometry of the bands; St-Dev are indicated. D. NIH-3T3 cells stably over-expressing L858R EGFR MT were treated as in A and EGFR was IPed as in B with either a528 or a4-2 Ab. Sample IBs of the IPs are shown for both EGFR (a2232) and IgG (amouse Ab), as indicated. The graphic in E. represents the averaged ratio between total EGFR (IPed with a528 Ab) and EGFR IPed with the a4-2 Ab, quantified by densitometry of the bands (as in C); St-Dev are indicated. doi:10.1371/journal.pone.0023240.g006 A549 cells were treated with 1 U/ml GO at 4, 16, 25, and 37uC or with 100 ng/ml EGF at 4uC. EGFR was IPed from protein extracts and IBed for total and Tyr-phosphorylated EGFR (Fig. 7). The data show that EGFR is activated by ox-stress only at 37uC whereas EGF activates EGFR even at 4uC (Fig. 7). Since H2O2 is generated by GO in a separate pre-incubation of the treatment media at 37uC (see methods), it is not likely that the lower treatment temperatures are interfering with H2O2 generation. Again, these results indicate that EGFR activation by ox-stress differs from its activation by the ligand, EGF, and could be dependent on membrane structure/fluidity. H2O2-induced activation of EGFR is inhibited by cholesterol up-take in the plasma membrane (PM) Pike and Casey [36] suggested that localization of EGFR to lipid rafts confers a functional inhibition of the receptor and that depletion of cholesterol releases EGFR from such inhibition, leading to an increase in basal EGFR phosphorylation. Consis- tently, it was shown later that cholesterol depletion from PM results in increased EGFR phosphorylation [20]. Given the possible involvement of membrane structure in EGFR activation observed under ox-stress (Fig. 3 and 7), we investigated the potential involvement of cholesterol in such EGFR activation. Others have shown before that disruption of lipid rafts by methyl-beta cyclodextrin (MbCD) treatment leads to EGFR activation and consequential distal signaling events [20]. There- fore, we first confirmed (Fig. 8A) that, indeed, cholesterol depletion from PM of A549 cells by MbCD treatment does activate EGFR, as was shown before [20]. Subsequently, A549 cells were treated with EGF or GO in the presence of 2 mM MbCD-cholesterol complex (CC) (prepared as described in ‘‘Material and Methods’’), known to cause a substantial uptake of cholesterol in the PM [37]. PLoS ONE | www.plosone.org 5 August 2011 | Volume 6 | Issue 8 | e23240