EL4 cells were lysed with glassbeads and the proteasome-enriched fraction was retrieved by consecutive ultracentrifugation steps as previously described [29] . Proteasome activity was inhibited with MG132 (50 mM). 10 mg of fraction protein were incubated with 0.2 mg of N-terminally HA-tagged ISG15VS in a total volume of 10 ml for two hours at room temperature to detect ISG15VSreactive proteases by anti-HA immunoblotting. The cleavage assay for ISG15-or SUMO1-branched peptides was performed at 37uC using 20 mg of total protein from the proteasome-enriched fraction and 5 ml of branched peptides in a total volume of 10 ml. HA-ISG15VS treated subcellular fractions were analyzed with a monoclonal anti-HA antibody (12CA5). Immunoblotting was performed as published [67] .