then harvested and total membranes separated. (A) Membranes were immunoblotted for SPT-LCB1, SPT-LCB2 and the a1 subunit of Na/K ATPase for total protein. The blot confirms the expression of SPT within MIN6b cells. (B) In vitro SPT activity was analysed as described previously [28]. Briefly, 80 mg of total membranes were taken and the SPT reaction buffer added (100 mM HEPES pH 8.3, 2.5 mM EDTA pH 7.0, 5 mM DTT, 3 50 mM Pyridaoxal Phosphate, 1 mM serine, 1 mCi [ H]-serine/ reaction and 10 mM AZ422 where indicated). Addition of palmitoyl-CoA initiated the assay (H2O substituted for palmitoyl- CoA in the control background sample). The reaction was allowed to proceed for 10 min at 37uC and terminated by the addition of 0.5 N NH4OH. Lipids were extracted using chloroform/methanol added to break the phases. NH4OH and sphinganine was added to act as a carrier for the lipid product generated. The resulting mixture was vortexed thoroughly and centrifuged. The upper aqueous phase was carefully removed and the lower organic phase, which contained the lipid products was washed twice with H2O. About 50–70% of the organic phase was vacuum-dried and the dried lipids reconstituted in 6.5:1:1 (v/v/v) chloroform/methanol/ acetic acid and radioactivity assessed. Bars represent mean 6 SEM from 3 separate experiments the asterisk signifies a significant difference between the indicated bars (P,0.05). (PDF) References 1. Corcoran MP, Lamon-Fava S, Fielding RA (2007) Skeletal muscle lipid deposition and insulin resistance: effect of dietary fatty acids and exercise. Am J Clin Nutr 85: 662–677. 2. Tuunanen H, Ukkonen H, Knuuti J (2008) Myocardial fatty acid metabolism and cardiac performance in heart failure. Curr Cardiol Rep 10: 142–148. 3. Anderwald C, Brunmair B, Stadlbauer K, Krebs M, Furnsinn C, et al. (2007) Effects of free fatty acids on carbohydrate metabolism and insulin signalling in perfused rat liver. Eur J Clin Invest 37: 774–782. 4. 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Briefly, during the 1 h glucose stimulation, cells were also incubated with 5 mM Oregon Green Bapta-1 probe (Molecular Probes, Invitrogen). After 1 h cells were washed 36 with KRH buffer and then viewed using a fluorescence microscope. Movies were taken over a 2–3 min time period but only the first 12 seconds are shown here to simplify the graph. Each time point is the average of 7 different cells but the error bars were removed, again for ease of view. Area under the curve analysis for each of the three conditions shown in the figure was performed using GraphPad prism and this analysis is presented in the table. (PDF) Author Contributions Conceived and designed the experiments: MW HH. Performed the experiments: MW KM. Analyzed the data: MW KM AM HH. Contributed reagents/materials/analysis tools: AM. Wrote the paper: MW HH. Contributed to the intellectual content/editing of the paper: MW AM HH. 17. 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