Rpn1-D517A reduces binding of Ddi1 in vitro. GST-fused Rad23, Dsk2, Ddi1 and GST alone (as a negative control) were incubated with either proteasomes affinity purified from rpn13Δ or rpn13Δ rpn1-V447H K484A D517A (VKD) cells. The binding reactions were immobilized on glutathione resin, which was then washed and extracted with SDS-PAGE sample buffer. An Rpt5 immunoblot (upper panel) and a commassie stain to confirm equivalent recovery of the GST fusion proteins (middle panel) is shown. Inputs were immunoblotted with anti-Rpt5 and are also shown (lower panel). A qualitatively similar result was obtained in two independent experiments.