HBx interacts with ERα and

Copyright information:Taken from "Hepatitis B virus X protein and the estrogen receptor variant lacking exon 5 inhibit estrogen receptor signaling in hepatoma cells"Nucleic Acids Research 2006;34(10):3095-3106.Published online 6 Jun 2006PMCID:PMC1475750.© 2006 The Author(s) () Interaction of HBx with ERα . A GST pull-down assay was performed using S-labeled ERα, and GST or GST-HBx. The bound proteins were subjected to SDS–PAGE followed by autoradiography. () Interaction of HBx with ERα . ERα and FLAG-tagged HBx or empty vector were co-transfected into HepG2 cells. Cell lysates were immunoprecipitated (IP) by anti-FLAG M2 monoclonal antibody (Sigma), and the precipitates were then immunoblotted (IB) with anti-ERα polyclonal antibody (Santa Cruz Biotech). () Interaction of endogenous HBx with ERα . Liver tissue extracts from an HBV positive patient were immunoprecipitated with either anti-ERα polyclonal antibody or preimmune control serum (Santa Cruz Biotech). The precipitates were analyzed by immunoblot using anti-HBx (Chemicon). () Effect of ERΔ5 on the interaction between HBx and ERα. HepG2 cells were co-transfected with 2 µg ERα, 4 µg FLAG-tagged HBx and increasing amounts of ERΔ5 (2 and 4 µg). Cell lysates were immunoprecipitated by anti-FLAG monoclonal antibody, and the precipitates were detected with anti-ERα polyclonal antibody. () Co-localization of HBx and ERα in HepG2 cells. Cells were transfected with EGFP-tagged HBx and RFP-tagged ERα or empty vector (RFP) as indicated, and were treated with 10 nM E for 24 h. The images were captured by confocal immunofluorescence microscopy. HBx localization is shown with EGFP (green) and ERα is seen with RFP (red). The nuclei were stained with DAPI (blue). Co-localization of HBx with ERα is shown in merged images. () Mapping of the ERα interaction region in HBx. A GST pull-down assay was performed using S-labeled ERα and GST-HBx(1-72), GST-HBx(73-120), GST-HBx(121-154), GST-HBx(1-143), GST-HBx(52-154) and full-length GST-HBx(1–154) or GST. Schematic diagram of the HBx deletion constructs used is shown at the top, the binding of ERα to different regions of HBx is demonstrated in the middle, and SDS–PAGE analysis of the purified GST-fusion proteins is shown at the bottom. Asterisks indicate the positions of the expected purified GST or GST-fusion proteins. () Mapping of the HBx interaction region in ERα. A GST pull-down assay was performed using full-length GST-HBx(1–154) or GST, and S-labeled full-length ERα (1