Figure 7. NEDP1 activity but not UBE2M activity is inhibited by oxidative stress.

1. In vitro de‐neddylation by NEDP1 is sensitive to oxidation. Poly‐NEDD8 reactions were prepared with NAE, UBE2M, and NEDD8 as in Fig 1E. NEDD8 reactions were stopped by the addition of NAE inhibitor MLN4924 (3 μM). NEDD8 reactions were incubated with GST‐NEDP1 at 30°C for the indicated amount of time under reducing (50 μM DTT) or oxidizing conditions (50 μM DTT + 150 μM H₂O₂). Reactions were stopped by the addition of LDS sample loading buffer and resolved by SDS–PAGE before processing for Western blot analysis with the indicated antibodies.
2. In vitro neddylation reactions with UBE2M are insensitive to H₂O₂. Poly‐NEDD8 reactions were prepared with NAE, UBE2M and NEDD8 as in Fig 1E. Before reactions were started, increasing concentrations of H₂O₂ (as indicated) were added to the NEDD8 reactions. Subsequently, the reactions were incubated at 30°C for the indicated amount of time. Reactions were stopped by the addition of LDS sample loading buffer and resolved by SDS–PAGE before processing for Western blot analysis with the indicated antibodies.