Figure 6
HBx induces a hepatocyte-specific BST-2 accumulation.
(a) The interaction between BST-2 and HBx was analyzed by the protein two-hybrid assay in 293T and L02 cells. Cells were transfected with 500 ng of pBIND(pB)-BST-2 IHA and 500 ng of pACT(pA)-HBx, along with 500 ng of pG5luc vector as indicated. The expression of pBIND-BST-2 IHA and pACT-HBx was detected by Western blotting using anti-HA and anti-HBx antibodies. The pBIND-Id and pACT-MyoD pair is a commercial positive control. (b) Firefly luciferase values in a) were detected and normalized by Renilla luciferase values. (c) 293T and L02 cells in 10-cm dishes were transfected with 2 μg of BST-2 IHA plasmid in the presence or absence of 2 μg HBx plasmid. The cell lysates were separated by cellular fractionation. The samples were analyzed by Western blotting using a mixture of anti-HA and anti-HBx monoclonal antibodies to detect BST-2 and HBx on a same blot. (d) HeLa or HepG2 cells on coverslips, transfected with 500 ng of LHBs-Flag or Flag-HBx expression vector and 100 ng of BST-2 IHA plasmid, were fixed and double-stained with a mouse anti-Flag antibody (secondary Ab: Alexa 488) and a rabbit anti-HA antibody (secondary Ab: PE). Images were taken under a Zeiss LZM710 confocal microscope. Scale bars: 20 μm. (e) The co-localization coefficient of HBV protein and BST-2 in d) is shown. At least 20 independent images were examined in each sample and the most representative cells are shown. (f) The diameters of the BST-2-localized cellular components in d) were measured with Image-Pro Plus 6.0 and are shown (n = 50). *P < 0.05. These experiments were performed three times and the most representative data are shown.
