Inc. and used as previously described [41]. The results of a BLAST search of all siRNA sequences revealed no significant homology with any other sequences in the database program. Construction and Preparation of GST Fusion Proteins GST fusion proteins of PLD1 were generated as previously described [41]. The individual fragments of PLD1 were then amplified by PCR using specific primers. They were digested with the restriction enzymes EcoRI and XhoI and ligated into pGEX- 4T1 vector. Next, E. coli BL21 cells were transformed with individual expression vectors encoding the GST fusion proteins and expression was then induced by culture in the presence of 0.5 mM isopropyl-b-D-thiogalactopyranoside (IPTG) at 25uC for 4 h. After harvesting cells, GST fusion proteins of PLD1 were purified as previously described [41]. Co-Immunoprecipitation HEK-293 cells were cultured, harvested and lysed with PLD assay buffer (50 mM Hepes/NaOH, pH 7.4, 3 mM EGTA, 3 mM CaCl2, 3 mM MgCl2 and 80 mM KCl) containing 1% Triton X-100, 1% cholic acid, 1 mM PMSF and protease inhibitor cocktail. After brief sonication, lysates were centrifuged at 100,0006g for 30 min and the supernatants (2 mg) were then incubated with anti-PLD-conjugated protein A Sepharose for 4 h. Co-immunoprecipitated complexes were washed three times with the same buffer and then analyzed by SDS-PAGE. The protein concentration was determined by Bradford assay. Immunoblot Analysis Immunoblot analysis was performed as previously described [42]. Briefly, proteins were denatured by incubation at 95uC for 5 min in Laemmli sample buffer. The denatured proteins were then separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking in TTBS buffer (10 mM Tris/HCl, pH 7.5, 150 mM NaCl and 0.05% Tween 20) containing 5% skimmed milk powder, the membranes were incubated with individual monoclonal or polyclonal antibodies and subsequently reincubated with either anti-mouse or anti-rabbit IgG coupled with horseradish peroxidase. Detection was performed using an enhanced chemiluminescence (ECL) kit according to the manu- facturer’s instructions. Measurement of PLD Activity in Cells PLD activity was assayed by measuring the formation of phosphatidylbutanol (PBtOH), which is the product of PLD- mediated transphosphatidylation, in the presence of 1-butanol [12]. Cells were labeled with [3H]myristic acid (10 mCi/ml) for 4 h and then washed twice with PBS. The labeled cells were maintained in glucose free-DMEM for 2 h at the indicated glucose concentrations and then incubated with 0.4% 1-butanol for 5 min. Next, cells were harvested into 0.8 ml of methanol and 1 M NaCl (1:1) and then mixed with 0.4 ml of chloroform. After vortexing, the samples were centrifuged at 15,0006g for 1 min, the organic phase was dried and the lipids were separated using Silica Gel 60 TLC plate. Finally, the plates were developed with ethyl acetate/trimethylpentane/acetic acid (9/5/2 = v/v/v). The amount of [3H]phosphatidyl-butanol was expressed as a percent- age of the total [3H]-lipid to account for differences in cell labeling efficiency. In Vitro Phosphorylation Assay AMPK was incubated with purified PLD1 (100 ng), GST- truncated PLD1 fusion mutants (1 mg) or precipitate complexes at PLD1 Regulation by AMPK 37uC for 15 min in kinase buffer (50 mM Tris/HCl, pH 7.4, 1 mM dithiothreitol, 0.02% Brij-35 and 10 mCi of [c-32P]ATP (3,000 Ci/mmol)) in the presence of AMP (200 nM). An in vitro phosphorylation assay was then performed as previously described [29,43]. The assay was terminated by the addition of 15 ml of 26SDS sample buffer and boiling of the samples for 5 min. The sample was then subjected to SDS-PAGE and exposed to photographic film for autoradiography. The protein concentration was determined by Bradford assay. In Vivo Phosphorylation Assay Initially, 16107 HEK-293 cells/100-mm dish were incubated with 3 mCi of [32P] orthophosphate in 5 ml of phosphate-free DMEM for 5 h at 37uC. Cells were then washed with PBS and treated with 50 mM 2-DOG for 10 min. Next, cells were washed with ice-cold isotonic buffer containing phosphatase inhibitor (30 mM NaF, 1 mM Na3VO4 and 30 mM Na4O7P2) and then lysed with 1 ml of lysis buffer (10 mM Tris/HCl, pH 7.5, 1 mM EDTA, 0.5 mM EGTA and 10 mM NaCl) containing 1% Triton X-100, 1% cholic acid, protease inhibitors (0.5 mM phenyl- methylsulfonyl fluoride, 1 mg/ml leupeptin, 5 mg/ml aprotinin) and phosphatase inhibitors (30 mM NaF, 1 mM Na3VO4 and 30 mM Na4O7P2). After centrifugation (15,0006g for 15 min), equal amounts of soluble extract were incubated with 2 mg of anti- PLD antibody and 30 ml of immobilized protein A. The immunoprecipitated proteins were then separated by SDS-PAGE, transferred to nitrocellulose membranes and exposed to photo- graphic film for autoradiography. The protein concentration was determined by Bradford assay. Determination of 14C-Glucose Uptake in Cells C2C12 cells (26105) or L6 GLUT4-myc myoblast cells (26105) were seeded in a 6-well plate. After differentiation of these cells, no significant differences of cell numbers were observed in each well. Morphological changes were confirmed to determine the differ- entiation. There was no significant cell death or proliferation during 14C-glucose uptake assay. 14C-glucose uptake was deter- mined as previously described [26]. Briefly, the differentiated C2C12 cells or L6 GLUT4-myc myotube cells were serum-starved for 2 h prior to the assay. Cells were then washed twice with Krebs-Henseleit buffer (NaCl 118, NaHCO3 25, KCl 4.6, MgSO4 1.2, KH2PO4 1.2 and CaCl2 2.5 mmol/l) and stimulated with or without the indicated agents. After incubation with 0.1 mCi/ml 2- deoxy[14C-glucose] at room temperature for 20 min, the uptake of 14C-glucose was terminated by washing cells three times with ice- cold PBS. Cells were then solubilized with 0.5 N NaOH and 0.1% SDS and their radioactivities were detected by scintillation counting. Each value was normalized to the total protein concentration. After the 14C-glucose uptake assay, the protein concentration was measured by Bradford assay and no significant differences were observed among samples. All assays were performed in duplicate. Measurement of the Cell Surface GLUT4 in Differentiated L6 GLUT4-myc Myotube Cells L6 GLUT4-myc myoblast cells were seeded at 26104 per well in a 24-well plate. After differentiation of these cells, no significant differences in cell numbers were among wells. There was also no significant cell death or proliferation observed during the cell surface GLUT4 assay. Quantitative analysis of the GLUT4 on the cell surface was measured by an antibody coupled-colorimetric absorbance assay, as described previously [44]. Briefly, after stimulation, cells were exposed to anti-myc antibody (1:100) for PLoS ONE | www.plosone.org 11 March 2010 | Volume 5 | Issue 3 | e9600