Figure 3

N-Glycosylation pattern of L protein deletion variants. Huh7 cells were infected at MOI 10 with rSFV encoding 1-48preS/S, G2A 1-48preS/S and 1-48preS/S₀. Twenty hours after infection cells were lysed with lysis buffer containing 0.5% Triton X-100, 150 mM NaCl, 50 mM Tris–HCl pH 7.5, 2 mM EDTA and 1 μg/ml phenylmethanesulfonylfluoride. Five-hundred U of PNGase F were added to 10 μl of Huh7 cell lysates diluted in reaction buffer (50 mM sodium phosphate buffer, pH 7.5) containing 1% NP40, and incubated for 1 h at 37°C. To perform the reaction under denaturing conditions, glycoprotein denaturing buffer was added to 10 μl of cell lysates and incubated for 10 min at 100°C, whereas for digestion under native conditions denaturing buffer was omitted. After separation by SDS-PAGE, proteins were transferred to a Hybond-P membrane in a semi-dry electro blotter. The membrane was reacted with MAb MA18/7, followed by goat anti-mouse antibodies conjugated with horseradish peroxidase. “+”, treated with PNGase F; “-”, untreated.