HBV Pol inhibits the activities of IKKs via interaction with Hsp90β.

<p><i>A</i>. HEK 293 T and HepG2 cells were transfected separately with 0.8 µg of the pCMV-flag-2B empty vector as a control or pCMV-flag-Hsp90β expressing the Hsp90β protein. At 24 h posttransfection, cell extracts were prepared, and the expression of the plasmids was determined using an anti-Hsp90β antibody. <i>B</i>. HBV Pol interacts with Hsp90β. 293 T cells were transfected with either the empty vector (pAHC) or pAHC-Pol in parallel with pCMV-flag-Hsp90β. At 24 h posttransfection, cells were harvested and subjected to immunoprecipitation. One-twentieth of the total cell lysates used for immunoprecipitation was loaded as a positive control for Hsp90β. <i>C</i>. HepG2 cells were transfected with 5×pNF-κB-Luc, pTK-Renilla-Luc and other plasmids as indicated. At 24 h posttransfection, cells were harvested for analysis by luciferase assay. <i>D</i>. HepG2 and HepG2.2.15 cells were transfected with 5×pNF-κB-Luc reporting system and Hsp90β or the empty vector as control. At 24 h posttransfection, cells were harvested for analysis by luciferase assay. Luciferase activities correspond to the average of results from at least three independent experiments, and data are shown as means ± SE (*p<0.05). <i>E</i>. 293 T cells were transfected with pAHC-Pol or the empty vector (pAHC) as a control. At 24 h posttransfection and after stimulation with TNF-α (10 ng/ml) for 15 min, cell extracts were immunoprecipitated with a control IgG1 (CTRL) or anti-NEMO (NEMO) monoclonal antibody. The presence of Hsp90, Cdc37, IKKα/IKKβ, and NEMO in the immunocomplexes was determined by immunoblotting.