Fig. 2. Ulp1 can remove Pmt3 from conjugates in S. pombe cell extracts. Recombinant Ulp1 was tested for deconjugating activity using wild-type (sp.011) S. pombe cell extract corresponding to 0.3 OD₆₀₀ units of cells per assay. Assays were analysed by SDS PAGE (12.5% polyacrylamide in (A) and 10% polyacrylamide in (B)) and western blotted with anti-Pmt3 antisera. (A) Lanes 1 and 2, no additions; lanes 3-6, 3.7 µg Ulp1. Lane 1, 0 minutes; lanes 2,6, 40 minutes at 30°C; lane 3, 5 minutes at 30°C; lane 4, 10 minutes at 30°C, lane 5, 20 minutes at 30°C. (B) Lanes 1,2, no additions; lanes 3-6, 3.7 µg Ulp1; lane 4, 10 mM iodoacetamide; lane 5, 10 mM NEM; lane 6, 0.5 mM PMSF. Lanes 7-9, 3.7 µg Ulp1 preincubated for 10 minutes on ice in the presence of 100 mM iodoacetamide (lane 7), 100 mM NEM (lane 8) or 5 mM PMSF (lane 9) before addition of cell extract. Lanes 2-9, incubated at 30°C for 30 minutes.