sucrose, 2 mM EDTA, 1 mM EGTA, 50 mM sodium fluoride, 100 mM sodium orthovanadate, 1%Triton X-100, 100 mM activated sodium orthovanadate, 10 mM b-mercaptoethanol, and protease inhibitor cocktail) as previously described [92]. Following homogenization, lysates were sheared using a 29G needle, incubated on ice for 30 min, and centrifuged at 16,0006g for 20 min. The supernatant was collected and protein concen- tration was assessed using Coomassie Plus Protein assay (Pierce). Lysates were treated with 16 pyronin sample buffer and subjected to SDS polyacrylamide gel electrophoresis (PAGE) in precast 4– 20% Tris-glycine mini gels (Invitrogen). After electrophoresis, proteins were electroblotted onto a polyvinylidene fluoride membrane with the use of a Hoefer Mighty Small Transphor unit at 400 mA for 90 min. Following transfer, the membrane was incubated in 5% BSA (Roche) and 0.1% Tween 20 in TBS (TBST) at room temperature for 1 h with shaking, followed by incubation with shaking overnight at 4uC with primary antibody diluted in 5% BSA in TBST. Membranes were washed with TBST and incubated with secondary peroxidase conjugated secondary antibody as per manufacturer’s instructions. Bands were developed using Pierce SuperSignal West FemtoTM maxi- mum sensitivity substrate. Blots were scanned using a Fluor-S Max MultiImager system (Bio-Rad, Hercules, CA). Quantification of bands was performed using Quantity One software (Bio-Rad). Monoclonal rabbit anti-EGFR, phospho-EGFR (Tyr1068), Akt, phospho-Akt (Ser473), ERK1/2, p-ERK1/2, STAT3, phospho- STAT3 (Tyr705), p70 S6kinase, and phospho-p70 S6kinase (Thr389) and monoclonal mouse phospho-tyrosine were pur- chased from Cell Signaling (Danvers, MA). Monoclonal mouse anti-ubiquitin and polyclonal rabbit anti-K, H, and N-Ras were purchased from Santa Cruz (Santa Cruz, CA). Affinity purified mouse anti-clathrin heavy chain and purified mouse anti-caveolin 1 were purchased from BD Transduction Laboratories (Bedford, MA). Peroxidase conjugated goat anti-rabbit IgG was purchased from Kirkegaard and Perry Laboratories (Gaithersburg, MD), and peroxidase conjugated goat anti-mouse IgG was purchased from Jackson ImmunoResearch (West Grove, PA). For detection of ganglioside GM-1, peroxidase conjugated cholera toxin B subunit was purchased from Sigma. Immunoprecipitation Cell lysates were incubated with rotation purified polyclonal rabbit anti-EGFR antibody (Millipore, Billerica, MA) overnight. The protein-antibody conjugates were then pulled down using protein-G conjugated Dynabeads (Invitrogen). Protein was eluted from the dynabeads using 26 pyronin buffer, and equal volumes of the samples were run on SDS-PAGE and Western blotted for EGFR, phospho-tyrosine, or ubiquitin. Lysates analyzed for ubiquitination of EGFR were treated with 5 mM N-ethylmalei- mide to prevent post lysis deubiquitination of EGFR [61]. Receptor Dimerization To assess EGFR dimerization, cells were treated as above. Following low serum incubation, cells were washed with ice-cold PBS prior to incubation with 0 or 25 ng/mL EGF on ice for 1 h. Cells were then washed with ice-cold PBS followed by incubation on ice for 20 min with 3 mM bis(sulfosuccinimidyl) suberate (BS3, Pierce), a non-permeable crosslinking reagent. In all experiments, a freshly prepared solution of BS3 was used. The crosslinking reaction was quenched by adding 250 mM glycine in PBS and further incubation on ice for 5 min. Cells were washed with PBS and homogenized as above. Protein concentration was measured and lysates were assessed by Western blotting for EGFR as described above. Regulation of EGFR Signal Transduction Fatty Acid Analysis To assess incorporation of DHA into phospholipids, membrane lipids were extracted with chloroform-methanol (2:1 vol/vol), and total phospholipids were separated by thin-layer chromatography with chloroform-methanol-acetic acid-water (90:8:1:0.8 vol/vol/ vol/vol). After transesterification, fatty acid methyl esters were quantified by capillary gas chromatography/mass spectrometry [93]. Total Internal Reflective Fluorescence (TIRF) Microscopy A plasmid containing the SH2 domain of Grb2 conjugated to YFP (Grb2-YFP) was generously provided by Alexander Sorkin, University of Colorado [94]. For TIRF experiments, cells were treated with fatty acids for 24 h prior to transfection. Cells were transfected using Amaxa nucleofection kit L (Amaxa, Basel, Switzerland) with 2.0 mg Grb2-YFP. Following transfection, cells were seeded at a density of 25,000 cells/well into MatTek (Ashland, MA) glass bottom 35 mm dishes in the presence of fatty acid. Approximately 32 h after transfection, cells were incubated in low serum media (0.5% FBS) containing fatty acid for 16–18 h prior to imaging. Images were acquired on a Zeiss TIRF Microscope system consisting of a Zeiss AxioObserver Z1 microscope (Carl Zeiss) equipped with a high resolution AxioCam MRm camera, argon laser, and AxioVision 4 software. An excitation wavelength of 514 nm was used, and emission was monitored at 537 nm. All images were collected using a 1006 objective (1.4 oil immersion) at 37uC and 5% CO2. To observe the translocation of Grb2-YFP to the plasma membrane, cells were stimulated with 100 ng/mL EGF, and images were collected every 5 sec. Images were collected using identical image acquisition parameters for all images within the experiment. Whole cell fluorescence intensity was performed on background- subtracted 16-bit images using Nikon Elements AR 3.2. Ras Activation Assay To assess the activation status of Ras, YAMC cells were treated for 72 h with fatty acid and incubated with low serum media (0.5% FBS) for the final 16–18 h. Cells were stimulated with 25 ng/mL EGF for 2 min, then harvested using Pierce cell lysis buffer. Activated (GTP-bound) Ras was subsequently isolated and assessed by Western blotting using the Pierce Active Ras Pull- Down and DetectionTM kit according to the manufacturer’s instructions. Additionally, Ras isoforms were detected by Western blotting the isolated GTP-bound Ras using isoform specific antibodies as described above. Biotinylation To assess cell surface EGFR and receptor internalization, cells were treated with fatty acids, incubated with low serum media and surface biotinylated using thiol-cleavable EZ-Link Sulfo-NHS-SS- Biotin (Pierce), 0.5 mg/mL, dissolved in PBS for 30 min on ice. Labeled cells were then rinsed two times with ice-cold PBS, and excess biotin was quenched with 60 mM iodoacetamide in PBS buffer for 5 min at 4uC. Select cultures were harvested at this step in order to quantify cell surface expression of EGFR. Cells were then washed three times with ice-cold PBS, followed by incubation at 33uC in prewarmed serum-free RPMI media for 5 min followed by stimulation with 25 ng/mL EGF for 0–30 min. Biotin groups remaining on the cell surface were then cleaved off by three 20 min washes with buffer containing reducing agent [100 mM MESNA (sodium-2-mercaptoethane sulfonate), 50 mM Tris (pH 8.6), 100 mM NaCl, 1 mM EDTA, and 0.2% BSA] at 4uC. Cells were then washed three times in ice-cold PBS and lysed in PLoS ONE | www.plosone.org 15 June 2012 | Volume 7 | Issue 6 | e39682