[26]. The CD98fl/fl IMCD cells were isolated from CD98hc floxed mice as described previously [63]. These cells are maintained in DMEM and 10% fetal calf serum. Established cells lines were transfected with a construct expressing full length human CD98 (hCD98) or vector only. Cells expressing hCD98 were selected using flow cytometry. IMCD cells expressing the Cless mutant (kindly donated by Dr. M. Palacin (Universitat de Barcelona)) [36,64] were generated by transfecting IMCD cells using Lipofectamine Plus (Invitrogen) with the Cless construct after which the cells were selected by adding Geneticin (G418 sulfate) (Invitrogen, Carlsbad, CA) at 1 mg/ml to the medium. Stable cell populations of cells expressing equal levels of Cless, CD98 and the mutants of CD98 were established utilizing flow cytometry under sterile conditions. Human Lat-1 cDNA was a generous gift from Dr. Francois Verrey (University of Zurich). Cells overexpressing Lat-1 were made by transfecting with the construct, after which they were selected with Zeocin (400 mg/ml) and cloned. Multiple clones were initially made and then transfected with CD98 or the mutant CD98 constructs as described above and sorted to equal levels of expression by flow cytometry. Cell Survival Studies Subconfluent monolayers (30–50%) of CD98fl/fl or CD98fl/fl IMCD cells reconstituted with hCD98 were infected with adeno- Cre for 16 hours. Lysates were isolated at 24 hr intervals following infection and run on 10–15% SDS gels. Immunoblots were probed with specific antibodies to human CD98 (Santa Cruz,C- 20) or mouse CD98 ( Santa Cruz, M-20) as well as cleaved Caspase-3 (Cell Signaling Technology, MA) and Actin as a loading control (Sigma-Aldrich). Amino Acid Transport The amino acid studies were performed as previously described [17]. The wild type or transfected IMCD cells were rinsed with warm Na+-free Hanks’ buffered salt solution (HBSS) (136.6 mm Choline Chloride, 5.4 mm KCl, 4.2 mm NaHCO3, 2.7 mm Na2HPO4, 1 mmCaCl2, 0.5 mm MgCl2, 0.44 mm KH2PO4, 0.41 mmMgSO4, pH 7.8), in which the sodium-containing salts were iso-osmotically replaced with choline, to remove extracellular Na+ and amino acids. Cells were equilibrated in warm choline- substituted HBSS for 10 min. The uptake of radiolabeled amino acids (2 mCi of [3H]Ile/ml) at 100 mmol/liter in 1 ml of choline- substituted HBSS was measured for 20 s at 37uC. Uptake of 3 [ H]Ile was linearly dependent on incubation time up to at least 3 min. Uptake was terminated by washing the cells rapidly four times with 1 ml/well of ice-cold HBSS. The cells were lysed overnight with 1 ml 0.2%SDS/2 ml NaOH. A 0.95-ml aliquot from each well was mixed with scintillation fluid, and radioactivity was quantified in a Beckman LS 6000SC liquid scintillation counter. The remaining 0.1 ml was analyzed for protein content using a BCA protein assay reagent. Cell Proliferation Assay 36103 cells were plated per well in 96-well plates and maintained in DMEM/F12 devoid of serum for 24 hours prior to the experiment. When studying serum-dependent prolifera- tion, 3H-Thymidine was added at 0.5 mCi/well in DMEM/F12 containing 1% FBS for up to 16 hours. When studying the effect of amino acids on proliferation, cells were incubated in DPBS containing glucose (3 g/L) and Ca++/Mg++ for 2 hours prior to the 8 hour 3H-Thymidine pulse. When needed, pretreatment with inhibitors was performed for up to an hour prior to the assay. The cells were then washed repeatedly with PBS then 10% CD98 Regulates Proliferation by Activating MAPKs Trichloroacetic Acid, solubilized with 1% SDS and radioactivity was measured using a liquid scintillation counter (Beckman, CA). Membrane Localization of CD98hc To verify the proper compartmentalization of CD98 in cells transfected with the deletion constructs, the cells were fractionated into Triton X-100–soluble and –insoluble fractions, as described previously [65]. Briefly, cells were scraped in cell lysis buffer (10 mM HEPES, pH 7.2, 1% Triton X-100, 100 mM NaCl, 2 mM EDTA supplemented with protease and phosphatase inhibitors), incubated at 4uC for 20 min, followed by centrifuga- tion at 10,000 6 g for 20 min. The resulting supernatant was considered the Triton X-100/detergent-soluble fraction. For the insoluble fraction, the pellet was dissolved in solubilization buffer (10 mM HEPES, pH 7.2, 1% SDS, 100 mM NaCl, 2 mM EDTA supplemented with protease and phosphatase inhibitors), and the proteins were released by sonication. The resulting preparation was incubated at 4uC for 20 min, followed by centrifugation at 10,000 6 g for 20 min. The resulting supernatant was considered the TX-100/detergent-insoluble fraction. 10 mg of each fraction was analyzed by Western blot using anti-hCD98 antibody. Amino Acid Deprivation Studie Cells were serum deprived for 24 hours, rinsed repeatedly with PBS then incubated for 2 hours in PBS containing Ca++/Mg++ and glucose at a concentration identical to that in DMEM/F12 (3 g/L). Serum free DMEM/F12 was then added to cells at different time points after which they were harvested for different assays. Pretreatment with inhibitors was performed prior to being treated with DMEM/F12. After establishing dose-response curves, inhibitors were used at the following concentrations: the MEK inhibitor U0126 10 mM, the p38 MAPK inhibitor SB203580 10 mM, the PI3K inhibitor LY294002 10 mM, the mTOR pathway inhibitor Rapamycin 100 nM and the mTOR kinase inhibitor PP242 2.5 mM. Immunoblotting Cells were serum starved overnight then stimulated with 10% FBS or subjected to amino acid deprivation as described above then lysed in cell lysis buffer (Cell Signaling Technology, MA) supplemented with protease and phosphatase inhibitors (Sigma, MO) at the time points indicated. After protein quantification total cell lysates (5–30 ug/lane) were run on 10–15% SDS gels, transferred to PVDF membranes (BioRad, CA) and blocked with 5% milk in TBS-T. Immunoblotting was performed with primary antibodies in 3%BSA in TBS-T then HRP-conjugated secondary antibodies in 5% milk/TBS-T and visualized using the ECL Plus western blotting detection system. Immunoprecipitation Cells overexpressing CD98 or CD98 deletion mutants as well as Flag-tagged human Lat-1 were lysed in a Triton X-100 based cell lysis buffer (Cell Signaling Technology, MA) supplemented with protease and phosphatase inhibitors. Pre-cleared protein lysates were incubated overnight with a human specific CD98 antibody (ATCC, Hybridoma cell line 4F2 C13) and Protein G sepharose beads (GE Healthcare) and run on a 10% SDS gel under both non-reduced and reduced (1 mM DTT) conditions. Immunoblots were probed with human specific CD98 antibody (Santa Cruz) as well as Flag antibody (Sigma, MO). PLoS ONE | www.plosone.org 10 June 2012 | Volume 7 | Issue 6 | e40026