Erasin interaction with ubiquilin-1, p97/VCP, and hHR23A. (A) Schematic drawing of ubiquilin-1. Ubiquilin-1ΔUBLΔUBA (178–428 aa) was used as the bait in a Y2H screen. (B) Yeast interaction assay of an erasin prey clone with different baits measured in five independent colonies showing the mean and SD (error bars). (C) GST pull-down assays of ubiquilin-1 (UBQ) fusion proteins with ³⁵S-radiolabeled erasin. (C, right) Schematic drawings of the GST constructs and a summary of their binding (+, binding; −, no binding). (C, left) Autoradiogram (top panel) and Coomassie-stained gel (bottom panel) of the pull-down experiment. (D) Coomassie-stained gel of purified proteins used for pull-down assays. The black line indicates that intervening lanes have been spliced out. (E) GST pull-down assays showing that erasin-His binds to GST-ubiquilin but not to GST or GST-hHR23A proteins. The input erasin, the recovered erasin, and GST proteins are shown. (F) GST pull-down assays showing that erasin, p97/VCP, and ubiquilin bind together in a trimeric complex. The input p97/VCP and erasin proteins and the recovered GST complexes (ponceau) are also shown. Arrowheads indicate the position of full-length GST-fusion proteins.