4 Lipid extracts obtained via the Folch procedure23 from 3 mL of LVPs or from 500 μL of each lipoprotein fraction

were separated by thin-layer chromatography (TLC) on Silica Gel G60 plates (Merck, Darmstadt, Germany) with hexane/diethyl ether/acetic acid (60/40/1, vol/vol) solvents. Phospholipid and triacylglycerol were scraped off the plate, and the molecular species composition of phospholipids separated by high-performance liquid chromatography (HPLC) on a silica-DIOL column (4 × 250 mm, Agilent 1100) was analyzed via electrospray ionization/tandem KPT330 mass spectrometry (Q-Trap 2000, Applied Biosystems). Phospholipid classes were eluted subsequently from HPLC as a function of the headgroup polarity using the solvent mixture hexane/isopropanol/aqueous selleck products ammonium acetate 5 mM 62.8/34.8/2.4 at the rate of 100 μL/minute. Experimental details have been discussed in recent reviews.24, 25 The method was set to detect the precursors (i.e., the parent phospholipids) of a characteristic fragment ion of each polar headgroup. Mass spectra were processed with Analyst software (v1.4.2, Applied Biosystems).

Assignment of the structure to mass peaks and deisotopization correction were performed with LIMSA software26 using a library prepared for the serum circulating lipids. Analysis of fatty acids in the triacylglycerol fraction separated by TLC (silica G25; solvent mixture hexane/methyl ether/formic acid: 80/20/2 vol/vol) was achieved by GC separation of methyl esters prepared by acid transmethylation according to Christie WW.27 Separation was achieved on a Carbowax 20 M capillary column (0.25 mm, 30 m, Quadrex) fitted on a Thermo-Electron 8000 GC chromatograph. A total of 20 μL of Y 27632 protein A–coated magnetic beads (Miltenyi Biotec) were incubated at room temperature with

1 mL of LDF in PBS with gentle rocking for 30 minutes. Samples were then passed through one magnetic column (Miltenyi Biotec) and washed with 2 mL of PBS and then with buffers of decreasing pH (successively, 300 μL of 0.1 M tris-acetate buffer pH 5.0, pH 4.0, and pH 3.0). Each collected fraction was immediately neutralized by addition of 0.1 N NaOH. Fractions eluted at pH 4.0 were used to stain the western blotting membrane. Immediately after preparation, LDFs or LVP fractions were collected in Laemmli buffer and denatured at 95°C for 5 minutes and kept at −20°C until analysis. Positive controls for E1 and E2 were obtained from lysates of cells expressing HCV glycoproteins or from supernatants of E1/E2-Caco2 differentiated cells,20 collected into Laemmli buffer, denatured, and conserved as described above. Samples were fractionated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane.