EGFR Activation under Oxidative Stress Figure 12. Proposed model of EGFR activation under ox-stress. A. Conventional activation/dimerization of EGFR upon stimulation by the ligand EGF. B. Ox-stress could cause a change in membrane structure/fluidity by increasing cellular ceramide levels, which could affect cholesterol distribution. This, together with possible direct effect of ox-stress on EGFR, induces, or stabilizes, a novel acquired active conformation of EGFR that is bound by active c-Src and caveolin-1 (Cav-1). Such aberrantly active EGFR does not dimerize ‘‘conventionally’’ and becomes resistant to TKI drug, while it traffics via caveolae to an unidentified peri-nuclear region, where it remains active. Please note: Cav-1 binding to EGFR under ox-stress was demonstrated by our group before [26]. doi:10.1371/journal.pone.0023240.g012 centrifugation and re-suspension) with the NP-40-lysis buffer were done prior to re-suspending the IPs in 50 ml of 26 loading dye (for SDS-PAGE, see below). Immuno-blotting (IB) 20–100 mg of total protein extracts or the IP samples were loaded into each well of the SDS-PAGE in the presence of a sodium dodecyl sulfate (SDS)/dithiothreitol (DTT) reducing loading dye (common concentrations/conditions – samples heated for 5 min. at 95uC). After SDS-PAGE separation the proteins were transferred to a nitrocellulose membrane and ‘‘blocked’’ with 5% skim milk in tris buffered saline with 0.05% tween-20 (TBST) for 120 min. or overnight. Primary Abs were incubated in 5% milk- TBST for 2 h at RT. Secondary Abs, either goat amouse- or goat arabbit-horseradish peroxidase (HRP) conjugated (Jackson Im- munoResearch), were incubated for 90 min. at RT at 1:10000 dilution in 5% milk-TBST. Bands were visualized by enhanced chemiluminescence (ECL, PIERCE). Extensive washes in TBST were done in between each step. Primary Abs used in this study for the IBs were: a2232 (aEGFR, Cell Signaling, 1:1000), ac-Src (Santa Cruz Biotech, 1:2000), a tyrosine Y416 phosphorylated (p-) c-Src (Cell Signaling, 1:2000), ap-Y20 (Santa Cruz Biotech, 1:3000), ap-Y1173 EGFR (Santa Cruz Biotech, 1:1000), ap- Y1086, ap-Y1068 and ap-Y845 EGFR (Cell Signaling, 1:1000). In vitro kinase assay Cells were scraped in a detergent-free buffer (50 mM Tris-Base 0.02% NaN3 1 mM EDTA in PBS) in the presence of protease and phosphatase inhibitors (Sigma), 1 mM NaF and Na3VO4, and then homogenized by several passages in a 30 gauge needle. Unbroken cells and nuclei were removed by a 5 min. centrifuga- tion at 5006g at 4uC; then, the homogenates were spun down by centrifugation at 60,0006g (Beckman ultra-centrifuge) for 30 min. to isolate the insoluble/membrane (pellet) fraction from the PLoS ONE | www.plosone.org 10 August 2011 | Volume 6 | Issue 8 | e23240