Figure 2

ORF2 protein increases the half life of cellular IκBα protein. (A) Huh7 cells were cotransfected with IKKβ and pSGI or pSGI ORF2 expression plasmids and pulse chased, as described in methods. Total IκBα was immunoprecipitated and samples were resolved on 12% SDS-PAGE and bands were detected by autoradiography. Band intensities were quantified using NIH Image program and graph was plotted assuming protein level at zero time to be 100%. In the graph, thin dotted line represents IKKβ transfected sample and solid line represents ORF2 and IKKβ cotransfected samples. Similar results have been obtained in 3 independent experiments. (B) Cytoplasmic (lane 1, 2) and nuclear (lane 3, 4) fractions of pSGI or pSGI ORF2 transfected cells were immunoprecipitated and subsequently western blotted with anti-p65 antibody (upper panel). A fraction of lysates were western blotted with calnexin and phospho c-Jun antibodies. Graph represents quantitation of p65 band intensities from the above image. Similar results have been obtained in 2 independent experiments. (C) Huh7 cells were transfected with expression plasmids: Lane 1, ORF2; lane 2, pSGI vector; lane 4 and 6, IKKβ; lane 3 and 5, ORF2 and IKKβ. Cell lysates were immunoblotted with phospho-IκBα, (ser 32), IκBα and calnexin antibodies. MG-132 was added 3 hours prior to cells harvesting. Graph shows quantitative representation of calnexin value normalized band intensities of phospho- IκBα and total IκBα from the above image. Similar results have been obtained in 3 independent experiments. (D) pSGI vector or pSGI ORF2 (lane 2 and 3) transfected cell lysates were immunoblotted with phospho IKKα/β (ser 180/181) and calnexin antibodies. 2 μg & 4 μg denotes the respective amount of DNA transfectedh.