FIG. 3.

Substrate specificity of Avp. (a) Cleavage of tetra-Ub and pro-ISG15 by recombinant Avp in the presence of pVIc peptide. Reactions were stopped at different times with NEM (50 mM) and analyzed by Western blotting with rabbit anti-Ub and anti-ISG15 antibodies. Where indicated, Avp was pretreated with the inhibitors LaggH (1 μM) or Ubal (1 μM) for 5 min at 4°C. In one case the activating peptide pVIc was omitted from reaction mixture. (b) Chromatographic analysis of Avp species on unoS column: Avp alone (I), Avp incubated with pVIc for 15 min (II), and Avp incubated with pVIc and Ubal for 30 min (III). (c) Cleavage of Ub-GST and Ub-pVIc-GST fusion proteins by Avp. The reaction conditions were as for Fig. 3a. The Avp cleavage sites are indicated in bold, whereas the pVIc-derived sequence is in italics. The anti-GST MAb B-14 and rabbit anti-Ub antibody were used for Western blotting.