Cul1 deneddylation is mediated specifically by CSN. a 1 nM His-6-Cul1/ROC1 was pre-neddylated for 30 min at 30°C; and for 20 min more, at 30°C, following the addition of elevated volumes of HeLa cells extract. Lanes
5–6 represent equivalent volumes of HeLa cells extract blotted with anti-Cul1 antibody; this compares the resultant fraction of endogenic Cul1 with its neddylated conjugate, to the extract blotted without the addition of His-6-Cul1/ROC1. Same deneddylation activity was achieved while using reticulocyte lysates as a source for COP9/Signalosome under the same conditions; the image was cropped since most of the work was conducted with HeLa cells extract due to technical reasons and the complete blot is provided as an additional data file termed Additional file 1. b 10 nM His-6-Cul1/ROC1 were pre-neddylated for 30 min at 30°C, followed by the addition of elevated volumes of HeLa cells extract, and by preparations that were either immunodepleted from JAB1 or that had undergone the same procedure without the antibody itself, as discussed in the methods section. The incubation time with the extract and its parallel derivatives was 20 min at 30°C. c 3 nM His-6-Cul1/ROC1 was pre-neddylated for 30 min at 30°C, followed by the addition of an elevated concentration of purified CSN for 10 min at 20°C. The first part of the image was cropped since it tested different conditions for neddylation and is not relevant to the data exemplified regarding the deneddylase activity of the purified CSN. The complete blot is provided as an additional data file termed Additional file 2.