Filipin blocks generation of sHJV but does not alter the glycosylation of cell-surface HJV. (A) Levels of cellular HJV were measured in tTA-HJV-HepG2 cells in the presence or absence of filipin and cycloheximide (CHX). Cells were grown in 6-well plates with 2 μg/mL doxycycline to induce HJV expression. Cells were treated for 2 hours in serum-free medium with or without cycloheximide (100 μg/mL) and filipin (10 μg/mL). One-half of the cell lysates (Lys) and one-fourth of the conditioned medium (CM) were subjected to Western analysis with anti-HJV and anti–β-actin or anti-Tf antibodies. Cells transfected with an empty vector (C) were used as a control for antibody specificity. (B) Analysis of cellular and cell-surface HJV by Endo H and PNGase F digestion in cells treated with filipin. Cells were treated as in panel A. For the cell lysate, 20% of a 60-mm dish was used. For the cell-surface HJV, biotinylated cell surface protein from approximately two-thirds of a 60-mm dish was used for each sample.