FIG. 4.

NESI interacts with HDAg-L in mammalian cells. (A) Coimmunoprecipitation of HDAg-L and HA-NESI protein. HepG2 cells were cotransfected with the expression plasmid of HA-NESI fusion protein (pcDNA3.1-HA-NESI) and a plasmid representing HDAg-S (pECE-d-SM), HDAg-L (pECE-d-BE), or HDAg-L(P205A) mutant protein [pECE-d-BE(P205A)]. Cell extracts were prepared 2 days posttransfection and subjected to immunoprecipitation with rabbit polyclonal antibodies specific for HA tag, followed by Western blot analysis with chicken antibodies against HDAgs as indicated. In addition, direct Western blot analysis was performed to serve as a control of the expression levels of HDAgs. (B) Specific interaction between HDAg-L and endogenous NESI. HepG2 cells were transfected individually with the expression plasmid of Flag-HDAg-L (pCMV-Tag2C-HDAgL), Flag-HDAg-S (pCMV-Tag2C-HDAgS), and the control plasmid pCMV-Tag2C. Immunoprecipitation was performed with M2-agarose beads specific for the Flag tag, and Western blot analysis was performed with rabbit antibodies against NESI and HDAgs as indicated.