Fluorescently labeled, photo-converted Ub(n)-FOLD was incubated with 26S proteasomes, Cdc48-UN, the proteasome cofactors Rad23, Dsk2, or Ddi1 (UBA-UBL proteins), and the Cdc48 cofactors Shp1, Ubx2, or Ubx5 (UBA-UBX proteins), as indicated. After 60 min, the samples were analyzed by SDS-PAGE and fluorescence scanning. The percentage of substrate degraded was quantified by determining the fluorescence in peptides (after background subtraction) and comparing it with the total fluorescence in each lane of the SDS gel. The experiment was performed in triplicates and the means and standard deviations are shown.
