Fig. 3

Reconstitution of ATP with AMP-PNP does not inhibit Cul1 deneddylation. a 3 nM His-6-Cul1/ROC1 was pre-neddylated for 30 min at 30°C, with either ATP or AMP-PNP, then followed with an addition of 100 nM purified CSN, on a time plot of 5–10 min at 20°C. Part of the image was cropped since the experiment also tested the use of CSN after the addition of ATP-trap mix with either ATP or AMP-PNP as was exemplified and discussed in Fig. 2 (regarding the use of ATP). Those parts of the experiment are not relevant for the question of CSN hypothetic autophosphorylation. The complete blot is provided as an additional data file termed Additional file 6. b 10 nM His-6-Cul1/ROC1 was pre-neddylated for 30 min at 30°C with AMP-PNP. Then 100 nM purified CSN was added at 20ºC (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).