stream signaling. Further understanding of the relationship between plasma membrane composition and receptor organiza- tion is required to fully elucidate the regulation of cell signaling. The interactions between cellular membranes and signaling proteins are emerging as important for mediation of cellular signaling [88,89]. Indeed, cell surface spatio-temporal organiza- tion is fundamentally dependent on the targeting of proteins to specialized membrane domains, as well as the dynamics of these protein-membrane associations. Ultimately, the intimate relation- ship of signaling proteins and membranes enables intricate control and refinement of biological processes. Based on the work presented here, fatty acids, specifically DHA, play a central role in regulating signal transduction by altering membrane interaction dynamics. Materials and Methods Cell Culture Young adult mouse colonic (YAMC) cells, conditionally immor- talized colonocytes, were originally obtained from R.H. Whitehead, Ludwig Cancer Institute (Melbourne, Australia). Both wild-type and EGFR2/2 isotype YAMC cells were utilized. YAMC cells (passages 12–17) were cultured under permissive conditions, 33uC and 5% CO2 in RPMI 1640 media (Mediatech, Manassas, VA) supple- mented with 5% fetal bovine serum (FBS; Hyclone, Logan, UT), 2 mM GlutaMax (Gibco, Grand Island, NY), 5 mg/mL insulin, 5 mg/ml transferrin, 5 ng/ml selenious acid (Collaborative Bio- medical Products, Bedford, MA), and 5 IU/mL of murine interferon-c (Roche, Mannheim, Germany). Select cultures were treated for 72 h with 50 mM fatty acid [DHA, linoleic acid (LA, 18:2n26), arachidonic acid (AA, 20:4n26), or eicosapentaenoic acid (EPA, 20:5n23); NuChek, Elysian, MN] complexed with bovine serum albumin (BSA). Where indicated, DHA treated cultures were washed three times with PBS followed by incubation with untreated media or LA for an additional 40 h. In select cultures, for the final 16–18 h, complete media was replaced with low-serum (0.5% FBS) media. Cells were then stimulated with 0–25 ng/mL recombinant mouse EGF (Sigma, St. Louis, MO) and harvested. In select cultures, cells were incubated with the ERK1/2 inhibitor U0126 (Invitrogen, Grand Island, NY) at 10 mM for 2 h prior to stimulation with EGF. Lipid Raft Isolation To determine the localization of EGFR within the plasma membrane, YAMC cells were treated with fatty acid and incubated with low serum media as above. Detergent-free lipid raft-enriched fractions were isolated as previously described [57,58]. All steps were performed at 4uC. YAMC cells grown in 12 T-175 flasks per treatment were harvested with trypsin-EDTA (Gibco) and pelleted at 2006g for 5 min. The pellets were resuspended in Buffer A (250 mM sucrose, 1 mM EDTA, 20 mM tricine, 100 mM activated sodium orthovanadate, 40 mL/mL protease cocktail, pH 7.8) at 16107 cell/mL. Cells from each treatment were pooled and lysed by two rounds of rapid freeze (280uC) and thaw (37uC). Cell lysates were then centrifuged at 10006g for 10 min, and the supernatants were retained. Cell pellets were resuspended in Buffer A and homogenized as above, and the centrifugation step was repeated. The resulting supernatants were pooled into a post-nuclear supernatant (PNS). The PNS was layered on top of 30% Percoll (Amersham, Pittsburg, PA) in Buffer A and centrifuged at 84,0006g for 30 min in a Beckman SW28 rotor. The plasma membrane fraction was collected and sonicated 3 times (50-J bursts) with 2 rapid pulses each time. The samples were then Regulation of EGFR Signal Transduction mixed with OptiPrep (Accurate Chemical and Scientific Corp, Westbury, NY) in Buffer A (final concentration 23%), overlaid with a 6 mL linear 20 to 10% OptiPrep gradient, and centrifuged in a Beckman SW41Ti rotor at 52,0006g for 90 min. The top 5 mL of the gradient was collected and combined with 4 mL of 50% OptiPrep in Buffer A. An aliquot of the denser membrane band (HDM) was collected. The 9 mL fraction was overlaid with 1 mL of 15% Optiprep in Buffer A and 0.5 mL of 5% Optiprep in Buffer A and was centrifuged at 52,000 g for 90 min in a Beckman SW41Ti rotor. A lipid raft/ caveolae-enriched membrane fraction (LR) was collected from the 5/15% interface, and a membrane fraction defined as the intermediate fraction (IDM) was collected at the bottom of the 15% layer. Slide-a-lyser cassettes (Pierce, Rockford, IL) were used to dialyze samples overnight in dialysis buffer (1 mM EDTA, 20 mM tricine, pH 7.8). Samples were placed into 1.5 mL Eppendorf tubes and centrifuged in a SpeedVac System to 1/3 the original volume to concentrate. Protein concentration was measured with Coomassie Plus Protein assay (Pierce), and fractions were subjected to SDS-PAGE and Western blotting as described above. Colocalization The plasmid containing RFP conjugated with a truncated H- Ras composed of the C-terminal 9 amino acid targeting domain (RFP-tH) was a generous gift from Ian Prior, Univ. of Liverpool [90]. The plasmid containing full length human EGFR conjugated to GFP containing a point mutation of A206K in the GFP sequence to prevent GFP dimerization (EGFR-mGFP) was a kind gift from Hung-Jun Liao, Vanderbilt University [91]. Cells were seeded at a density of 1.06105 cells per well into Lab-Tek II 2-well chambered coverglass slides (Nalge Nunc, Rochester, NY) 24 h prior to transfection and cultured in complete RPMI media containing 50 mM fatty acid. Cells were cotransfected with 0.3 mg RFP-tH and 1.5 mg EGFR-mGFP using Effectene (Qiagen, Valencia, CA) in media without fatty acid according to the manufacturer’s instructions. Transfection conditions were opti- mized to minimize the amount of DNA and lipofection reagent used to avoid nonspecific cytotoxicity. Four h after transfection, cells were washed and the media was replaced and supplemented with 50 mM fatty acid for 48 h. Cells were incubated in low serum media (0.5% FBS) with fatty acid for the final 16–18 h prior to imaging. Cells were imaged 48 h after transfection. Prior to imaging, cultures were washed with Leibovitz’s L-15 medium (Gibco) followed by addition of 1 mL of Leibovitz’s media (without serum) per well. Images were collected with a Zeiss 510 META NLO Multiphoton Microscope System consisting of an Axiovert 200 MOT microscope (Carl Zeiss Microimaging, Thornwood, NY) equipped with an argon laser, PMT, and LSM software. For EGFR-mGFP and RFP-tH, excitation wavelengths of 488 nm and 543 nm were used, and fluorescence emission was monitored at 530 nm and 590 nm, respectively. Images were collected in confocal mode with the pinhole set at 1 AU using a 406 objective (1.3 oil immersion lens) at room temperature. Identical acquisition parameters were used for all images within the experiment. Colocalization at the plasma membrane was analyzed by quantifying Mander’s colocalization coefficient for green (EGFR-mGFP) with red (RFP-tH) using Nikon Elements AR 3.2. Analysis was performed on background-subtracted 16-bit images. Western Blotting For Western blotting, cells were homogenized in ice-cold homogenization buffer (50 mM Tris-HCl, pH 7.2, 250 mM PLoS ONE | www.plosone.org 14 June 2012 | Volume 7 | Issue 6 | e39682