Direct inhibition of APP-BP1/UBA3 with MLN-4924 results in inhibition of CSN deneddylase activity. a 10 nM His-6-Cul1/ROC1 was pre-neddylated for 30 min at 30°C. Then 10 μg HeLa cells extract was added, on a time plot at 20°C (lanes 3–6); or, alternatively, MLN-4924 was added for 15 min at 30°C, followed by the addition of 10 μg HeLa cells extract, on a time plot at 20°C (lanes 8–11). In lane 1, incubation with MLN-4924 was prior to neddylation. b The same as a using 100 nM purified CSN instead of HeLa cells extract. Both images 5a, b were cropped and the lanes representing the end of the time plot (200 s) were omitted due to technical reasons. The complete blots are provided as additional data files termed Additional file 9 and Additional file 10, respectively.