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Fig. 4 | Cell Division

Fig. 4

From: COP9-Signalosome deneddylase activity is enhanced by simultaneous neddylation: insights into the regulation of an enzymatic protein complex

Fig. 4

Endogenic Cul1 deneddylation is enhanced during simultaneous neddylation. a 20 μg HeLa cells extract were incubated for 30 min at 30°C (lanes 27) or with the addition of ATP-trap mix prior to the incubation (lanes 813). Simultaneous ATP-depletion was performed in lanes 37 after 30 min incubation, on a time plot of 1.5–7.5 min at 30°C. Simultaneous neddylation was performed in lanes 913 after 30 min, by the addition of AMP-PNP, on a time plot of 1.5–7.5 min at 30°C. b 10 nM His-6-Cul1/ROC1 was neddylated at 30°C on a time plot of 2.5–30 min, without or with the addition of ATP-trap mix (lanes 26 and lanes 711, respectively). In lane 12, the neddylation mix was added to the reaction after pre-incubation with ATP-trap mix. c 10 nM His-6-Cul1/ROC1 was neddylated at 30°C using AMP-PNP on a time plot of 2.5–30 min, either directly (lanes 26) or after pre-incubation with ATP-trap mix (lanes 711).

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