Secretion of HCV Glycoproteins Figure 8. MTP activity inhibition decreases HCV envelope glycoprotein secretion by E1E2-Caco-2. E1E2-Caco-2 cells were cultured under differentiating condition for 11 days when medium was replaced by FCS free medium with the MTP inhibitor Cp 346084 at 0, 1 or 10 nM. MTP activity was measured on frozen lysates and expressed as described in the Materials and Methods section. (A) MTP activity in lysate of E1E2-Caco-2, 24 and 48 h after addition of Cp 346084. MTP activity was not completely abrogated by the addition of the inhibitor since its activity continued to increase during the inhibition period. However, there was a dose dependant inhibition of MTP activity by 1 and 10 nM Cp 346084. (B) Concentrations of secreted ApoB in the basal medium were measured by ELISA at 24 and 48 h after addition of 1 or 10 nM Cp 346084. ApoB secretion was inhibited in a dose dependant manner by the MTP inhibitor. (C) Secretion of HCV glycoprotein E1 and E2 in the basal medium was monitored by western-blotting with the E1-specific A4 and E2-specific H52 antibodies at 24 and 48 h after addition of 1 or 10 nM Cp 346084. Control cellular E2 protein was deposited in lanes (+). Amount of secreted E1 and E2 was quantified by densitometry as per cent of secreted glycoprotein in absence of inhibitor (upper panels). Cellular glycoprotein expression was monitored by western-blotting of cell lysates and was not modified by MTP inhibition (lower panel). doi:10.1371/journal.pone.0004233.g008 0.25 M sucrose) and 56.4% (wt/vol) (16.0 ml of 60% iodixanol, 0.34 ml of 0.5 M Tris-HCl, pH 8.0, 0.34 ml of 0.1 M EDTA, pH 8.0, and 0.34 ml 0.25 M sucrose) iodixanol solutions in thinwall centrifuge tubes (14689 mm, Beckman) using a two- chamber gradient maker. A sample of 1 ml lower compartment medium was applied to the top of 6 to 56% iodixanol gradients and centrifuged for 10 h in a Beckman Optima L100 XP ultracentrifuge at 41000 rpm and 4uC in an SW41 rotor. The gradient was harvested by tube puncture from the bottom and collected into 11 (1 ml each) or 22 fractions (0.5 ml each). The density of each fraction was determined by measuring the mass of 100 mL. Co-immunoprecipitation Co-immunoprecipitations were performed with mMACS Protein G Magnetic MicroBeads according to the manufacturer’s instruc- tions (Miltenyi Biotec, Bergisch Gladbach, Germany). 0.2 mg of anti-E2 H48 was added to 2 ml of the lower compartment medium before overnight incubation at 4uC. After one hour incubation with 20 ml of protein G microbeads, labelled immune-complexes were applied into a mMACS MS column. After one wash with PBS/BSA 0.2%, the retained immunoprecipitate was eluted in 100 ml of pre- heated (95uC) gel loading buffer. Western blot Samples preparation: Media or gradient fractions were collected in Laemmli buffer and immediately frozen at 220uC until analysis. Cell layers were briefly rinsed twice with ice-cold PBS (Invitrogen), scraped into 0.5 ml of lysis buffer (1% Triton X-100 and 5 mM EDTA in PBS) supplemented with 2% (v/v) protease inhibitor cocktail (P8340; Sigma–Aldrich), and immediately frozen at 220uC. Cell lysates were run through a small bore needle (25G 5/80) a few times. The protein concentration was determined using the Micro BCA Protein Assay (Perbio Science) with BSA as standard protein and samples were then diluted in loading buffer prior to fractionation by SDS-PAGE. Positive controls for ApoB100 and ApoB48 were obtained from purified human lipoproteins as follows. Plasma sample from a postprandial healthy PLoS ONE | www.plosone.org 10 January 2009 | Volume 4 | Issue 1 | e4233