Fig. 2

ATP-depletion resulted in the inhibition of CSN deneddylase activity. a 10 nM His-6-Cul1/ROC1 were pre-neddylated for 30 min at 30°C. Then 10 μg HeLa cells extract were added at 30°C (time plot: lanes 3–6); or alternatively, ATP-trap mix was added for 45 min at 30°C, followed by the addition of 10 μg HeLa cells extract at 30°C (time plot: lanes 8–11). b 3 nM His-6-Cul1/ROC1 was pre-neddylated for 30 min at 30°C. Then 100 nM purified CSN was added at 20°C (time plot: lanes 3–6); or alternatively, ATP-trap mix was added for 45 min at 30°C, followed by the addition of 100 nM purified CSN at 20°C (time plot: lanes 8–11). c 10 nM His-6-Cul1/ROC1 was incubated as described in b; time plots were for 1.5–7.5 min, and not for 5–40 min as in b. Blotting was directed against NEDD8, and not against Cul1, as described elsewhere.