Secretion of HCV Glycoproteins Figure 9. MTP activity inhibition decreases HCV envelope glycoprotein secretion by E1E2-HepG2 and E1E2-Huh-7.5. E1E2-HepG2 and E1E2-Huh-7.5 cells were cultured in standard conditions until 80% confluence when medium was replaced by FCS free medium with 0, 1 or 10 nM of MTP inhibitor Cp 346084 for two days. MTP activity was measured on frozen lysates and expressed as described in the Materials and Methods section. (A) E1E2-HepG2 and (B) E1E2-Huh-7.5. Upper panels, MTP activity in cells and apoB concentrations in medium in presence of the indicated inhibitor concentrations. Middle panels, quantification of secreted glycoproteins in presence of 1 and 10 nM MTP inhibitor by western blotting and densitometry as per cent of secreted glycoproteins in absence of inhibitor. Lower panels, cellular glycoprotein expression was not modified by the MTP inhibitor as shown by western-blotting of cell lysates. doi:10.1371/journal.pone.0004233.g009 donor was centrifuged for 4 h at 4uC and 543000 g with a TL100 ultracentrifuge (Beckman instruments). Upper low-density fraction was collected and 100-fold diluted in Laemmli buffer before denaturation. Positive controls for E1 and E2 were obtained from a lysate of cells expressing HCV glycoproteins. 20 mg of cell protein lysates, 40 ml of gradient fraction or 20 ml of immunoprecipitate were heated for 5 minutes at 95uC, fractionated by SDS-PAGE and transferred to a PVDF mem- brane. After an overnight incubation at 4uC in PBS-0.1% Tween 20 supplemented with 5% (w/v) non-fat milk powder, blots were probed with mAb against apoB, E1 or E2 diluted in PBS-0.1% Tween 20 (1:5000 for apoB, 1:1000 for E1 and E2 ) for 90 minutes followed by a peroxidase-conjugated goat anti-mouse antibody (Perbio Science; 1:5000 dilution for 1 h in the same buffer). Blots were developed with enhanced chemiluminescence reagents according to the manufacturer’s instructions (SuperSignal West Femto Maximum Sensitivity Substrate , Perbio Science). MTP Activity MTP activity was measured with the Research Assay Kit for MTP (Chylos Inc.) according to manufacturer instructions with minor modifications. Briefly, for kinetic study, cells in inserts were washed with ice-cold PBS, dryed and frozen at day 7, 9, 11 and 13, and stored at 220uC until MTP activity assay. Cells from a 4.2 cm2 insert were homogenized into 150 ml of 50 mM tris-HCl, pH 7.4, 50 mM KCl and 5 mM EDTA homogeniza- tion buffer. Cell lysates were run through a small bore needle (25G 5/80) twenty times. The assay was done in white microplates (Nunc GmbH, Wiesbaden, Germany). To the wells, we added 10 ml of cell homogenate, 5 ml of substrate vesicles mix and distilled water to make a final volume of 100 ml. Microplates were incubated at 37uC for 2 h before fluorescence reading using 460 nm excitation and 530 nm emission wave- lengths into a Wallac 1420 Victor2TM microplate Fluorimeter (PerkinElmer, Courtaboeuf, France). To determine background fluorescence value, vesicles fluorescence was measured in absence of cell lysate after 2 h incubation in the same conditions. Background value was then subtracted from each sample. Protein concentrations in cell homogenates were determined using a Bradford protein assay kit (Sigma-Aldrich) and results were expressed as units of fluorescence per mg of protein. PLoS ONE | www.plosone.org 11 January 2009 | Volume 4 | Issue 1 | e4233