Fig. 6. Erasin binds p97/VCP proteins primarily through its UBX domain. (A) Immunoprecipitation assays demonstrating that erasin forms a complex with p97/VCP. Lysates were prepared from HeLa cells that were either left untransfected (lanes 1 and 2), or transfected with a FL untagged erasin cDNA plasmid (lane 3), and used to immunoprecipitate erasin using the preimmune 141 serum (lane 1) or with anti-erasin antibody 141 (lanes 2 and 3). Lane 4 contains 1/10 of the lysate prepared from untransfected cells that was used for the immunoprecipitation shown in lane 1 and 2. After separation by SDS-PAGE, the immunoprecipitates were immunoblotted for erasin, p97/VCP and calnexin proteins (upper three panels, respectively). The bottom two panels show 1/10 the amount of total protein lysates from the same sets of cells immunoblotted for erasin, and p97/VCP. (B) Immunoblot demonstrating recombinant p97/VCP is pulled down by GST-erasin fusion protein but not by GST alone. (C) GST pull-down assays showing that erasin binds p97/VCP primarily through its UBX domain. In vitro translated ³⁵S-radiolabeled proteins generated from different erasin constructs (labeled; arrows indicate the major erasin translation product) were analyzed for binding FL p97/VCP GST-fusion protein by pull-down assays (upper panel, autoradiogram of the pull-down results; middle panel, 1/10 portion of the input sample; lower panel, Coomassie-stained gel of pulled down GST-p97/VCP-fusion protein). (D) Immunoprecipitation assays demonstrating that the UBX domain of erasin is important forming a complex with p97/VCP in cells. Lysates prepared from HeLa cells transfected with either FL Myc-tagged erasin or Myc-tagged erasin with deleted UBX domain (UBX) were used to conduct immunoprecipitations with a mouse anti-Myc antibody. The immunoprecipitates, together with 1/10 portion of the lysate, were immunoblotted with anti-p97/VCP (upper panel) and anti-Myc antibodies (lower panel).