FIG 10.

Involvement of protease activity in the degradation of RNF2 in HCV-infected cells or core-expressing cells. (A) HCV-infected and mock-infected Huh7OK1 cells were transfected with a plasmid encoding HA-tagged ubiquitin or the empty plasmid. The transfected cells were treated with 10 μM MG-132 or DMSO at 36 h posttransfection and harvested at 40 h posttransfection. RNF2, polyubiquitinated (polyUb) proteins, NS5A, and β-actin in cell lysates were evaluated by immunoblotting (left panels), while the proteins immunoprecipitated with an anti-HA antibody were evaluated by immunoblotting with an anti-RNF2 antibody (right panel). (B) HCV-infected cells and mock-infected cells were transfected with siRNA targeting E6AP mRNA, siE6AP1, or siE6AP2. The cells were harvested at 48 h posttransfection. The cell lysates were subjected to Western blotting. (C) Plasmids encoding the core protein and/or RNF2-HA were transfected into Huh7OK1 cells. The cells were treated with 10 μM MG-132 or DMSO at 36 h posttransfection and were then harvested at 40 h posttransfection. The cell lysates were subjected to Western blotting. The data shown in this figure are representative of three independent experiments.