Figure 3. Cyclic peptides inhibit the DUB cleavage of K48-linked di/tetra-Ub.

a) ^K48Ub₂ incubated with the K48-linked specific DUB OTUB1 for 1 hour resulted in cleavage to Ub₁. This reaction was inhibited by all cyclic peptides. b) ^K48Ub₂ incubated with the non-linkage-specific DUB USP2 resulted in cleavage to Ub₁, cleavage could be inhibited by all peptides. c) Ub4ix inhibits OTUB1 in a dose dependent manner, with an IC₅₀ approximately the same as the substrate concentration (^K48Ub₂ initially at 2 μM). d) Excess of Ub4ix (relative to ^K48Ub₂ substrate) was required to fully inhibit the activity of USP2 over a 1 hour incubation. e) ^K48Ub₄ incubated for 2 hours with OTUB1 resulted in cleavage to ^K48Ub₃, ^K48Ub₂ and Ub₁. In the presence of cyclic peptides, cleavage to ^K48Ub₃ occurred, but further cleavage was inhibited. f) Similar for OTUB1, ^K48Ub₄ incubated for 1 hour with USP2 could cleave to ^K48Ub₃, ^K48Ub₂ and Ub₁, and this cleavage was inhibited by the cyclic peptides. In all experiments, equal amounts were loaded on SDS-PAGE gel, electro-blotted to nitrocellulose membrane, and probed with an anti-Ub antibody. Initial concentrations of ^K48Ub₂ or ^K48Ub₄ substrate were 2 μM in each case.