Analyzing HBV-Containing Autophagosomes

HepG2.2.15 cells containing transfected HBV full-length DNA [17 (link)] or HepG2 cells were treated with 200 nmol/L Bortizomib (Millennium pharmaceuticals, USA) alone or in combination with 100 nmol/L Rapamycin (Enzo Life Sciences, China) or 30 mmol/L NH₄Cl for 18 h, DRibbles containing autophagosomes were prepared from the culture media as described [13 (link),14 (link)]. Morphology analysis of HBV⁺ DRibbles was done under transmission electron microscopy. HBsAg in HepG2.2.15 cell lysates was detected by western blot analysis using anti-HBsAg antibody (Santa Cruz, Biotechnology, Inc., USA). LC3 in both HepG2.2.15 cell lysates and HBV⁺ DRibbles was determined by western blot analysis using the polyclonal LC3B antibody (Cell Signal Technology, USA,1:1000), the HRP-labeled goat anti-rabbit IgG secondary antibody (1:5000) and a chemiluminescence kit (Multisciences Biotech Co., Ltd., China). Levels of HBsAg and HBeAg in HBV⁺ DRibbles were measured by ELISA (Shanghai Kehua Bioengineering Co., Ltd., China).