RIPA buffer (20 mM Tris, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1% sodium deoxycholate, 1% SDS, and 1% Triton X- 100) containing protease and phosphatase inhibitors. The lysates were clarified by centrifugation at 16,0006g for 20 min. Protein concentration was assessed using BCA protein assay (Pierce). Biotinylated EGFR was captured on streptavidin ELISA plates (NUNC IMMOBILIZER Streptavidin C8) from the cell lysates diluted to 5 mg/mL total protein in PBS containing 0.5% Tween 20, pH 7.3 (PBST), during a 2 h incubation at room temperature on a shaker. Plates were then washed three times with PBST, incubated with anti-EGFR antibody (Santa Cruz) (2 mg/mL) for 2 h at room temperature, washed, and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The plates were subsequently washed three times in PBST before adding color substrate (R&D Systems, Minneap- olis, MN) for 5–10 min. Color development was stopped by addition of an equal amount of 4 M H2SO4 and analyzed at 450 nm. Cell Proliferation YAMC cells (both wild-type and EGFR2/2) were treated with fatty acids for 24 h prior to being seeded into a 96-well plate at a density of 1.06105 cells per well. Cells were cultured in complete media supplemented with fatty acids for an additional 48 h. Additionally, wild-type YAMC cells were transfected using nucleofection (Amaxa kit L) with a constitutively active form of H-Ras (H-RasG12V-GTP), provided by Dr. Ian Prior [95], prior to being seeded into a 96 well plate and cultured for 48 h. Cells were then washed with PBS and cell proliferation was measured using CyQuant cell proliferation assay (Molecular Probes, Grand Island, NY) according to the manufacturer’s instructions. Mice Female C57BL/6 wild-type mice were used. All procedures followed guidelines approved by the U.S. Public Health Service and the Institutional Animal Care and Use Committee at Texas A&M University. This study and all animal protocols and procedures were approved by the Institutional Animal Care and Use Committee at Texas A&M University (Permit number: AUP2010-079). All efforts were made to minimize suffering. Mice were maintained under barrier conditions and initially consumed a Teklad commercial mouse non-purified diet (Harlan, Indianapolis, IN) ad libitum. Mice were maintained for 15 weeks on a semi- purified defined diet that was adequate in all nutrients, containing 320 (g/kg diet) sucrose, 200 casein, 220 corn starch, 3 DL- methionine, 35 AIN 76 salt mix, 10 AIN 76 mineral mix, 2 choline chloride, 60 cellulose (Bio-Serv, Frenchtown, NJ), 150 corn oil References 1. Olayioye MA, Neve RM, Lane HA, Hynes NE (2000) The ErbB signaling network: receptor heterodimerization in development and cancer. EMBO J 19: 3159–3167. 2. 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Regulation of EGFR Signal Transduction (CO) (Dyets, Bethlehem, PA) or 115 vacuum-deodorized Menha- den fish oil (FO) (Omega Protein, Houston, TX) plus 35 CO. The animals were provided the defined diet 2 weeks prior to an injection of azoxymethane (AOM; 7.5 mg/kg body weight). One week after the AOM injection, mice were exposed to 3 cycles of 1% dextran sulfate sodium (DSS) for 4 d followed by 17 d of recovery. Mice were sacrificed 6 weeks after the last DSS treatment (13 weeks post AOM injection) by CO2 asphyxiation. Colon lesions were mapped and excised prior to colonic mucosa isolation by scraping, and the mucosa was snap frozen in liquid nitrogen and stored at -80uC until homogenization. Lesions were fixed in 4% paraformaldehyde, embedded in paraffin, stained with hematoxylin-eosin, and evaluated in a blinded manner by a board- certified pathologist. For immunoblotting, mucosa was homoge- nized in ice-cold homogenization buffer as described above. For immunoprecipitation, mucosa was homogenized using Pierce ClassicTM IP lysis buffer according to the manufacturer’s instructions. Following homogenization, lysates were sheared using a 29G needle, incubated on ice for 30 min, and then centrifuged at 16,0006g for 20 min. The supernatant was collected. Protein concentration was determined using Coomassie Protein Plus assay (Pierce). Statistics The effect of 2 independent variables (treatment effects) was assessed using Student’s t-test. The effect of more than 2 independent variables (treatment effects) was assessed using the one-way analysis of variance test (ANOVA), and differences among means were evaluated using Tukey’s post-hoc test of contrast. P values ,0.05 were considered to be statistically significant. Acknowledgments We thank Alexander Sorkin (University of Colorado), Hung-Jun Liao (Vanderbilt University), and Ian Prior (University of Liverpool) for plasmids used in this study; Evelyn Callaway for help with the animal study and technical assistance. We also acknowledge the Texas A&M University Image Analysis Lab for its excellent resources and the expertise of Robert Burghardt. We are grateful to members of the Chapkin laboratory for helpful discussions and comments on the manuscript. Author Contributions Conceived and designed the experiments: HT RC. Performed the experiments: HT. Analyzed the data: HT. Contributed reagents/ materials/analysis tools: RC. Wrote the paper: HT RC. Performed imaging: HT RB. 7. Pike LJ, Han X, Gross RW (2005) Epidermal growth factor receptors are localized to lipid rafts that contain a balance of inner and outer leaflet lipids: a shotgun lipidomics study. J Biol Chem 280: 26796–26804. 8. Pike LJ (2006) Rafts defined: a report on the Keystone Symposium on Lipid Rafts and Cell Function. J Lipid Res 47: 1597–1598. 9. Chen X, Resh MD (2002) Cholesterol depletion from the plasma membrane triggers ligand-independent activation of the epidermal growth factor receptor. J Biol Chem 277: 49631–49637. 10. Pike LJ, Casey L (2002) Cholesterol levels modulate EGF receptor-mediated signaling by altering receptor function and trafficking. Biochemistry 41: 10315– 10322. 11. Pike LJ, Casey L (1996) Localization and turnover of phosphatidylinositol 4,5- bisphosphate in caveolin-enriched membrane domains. J Biol Chem 271: 26453–26456. 12. Pike LJ, Miller JM (1998) Cholesterol depletion delocalizes phosphatidylinositol bisphosphate and inhibits hormone-stimulated phosphatidylinositol turnover. J Biol Chem 273: 22298–22304. 13. Westover EJ, Covey DF, Brockman HL, Brown RE, Pike LJ (2003) Cholesterol depletion results in site-specific increases in epidermal growth factor receptor PLoS ONE | www.plosone.org 16 June 2012 | Volume 7 | Issue 6 | e39682