FIGURE 5.

Ulp1 liberates poly-Smt3 from a substrate chain. Purified 5xSmt3-GFP was incubated with a 1:2000 dilution of lysate containing MBP-Ulp1-FLAG. The lysate was diluted in reaction buffer. At the indicated time points, samples were withdrawn, and the reaction was stopped by the addition of SDS Laemmli buffer and subsequent boiling. Reaction mixtures were then separated by SDS-PAGE and subjected to Western blot detection of the FLAG tag. Extract-derived or unspecific bands are indicated by asterisks (*). The full-length substrate is indicated on the left-hand side of each blot, and cleavage products are indicated on the right. A, assay was performed on ice. B, assay was performed at 30 °C.