Figure 5.
DCNL1 activity is independent of its UBA domain and not altered by monoubiquitylation. Reconstitution of CUL5–RBX2 neddylation with recombinant purified NEDD8-E1, N-terminally acetylated UBE2F, and WT DCNL1 or DCNL1 variants. Side-by-side comparisons of CUL5 neddylation in the absence of DCNL1 (mock) and between DCNL1 and DAD-patch mutant of DCNL1 (A), and DCNL1 (PONY) (B), monoubiquitylated DCNL1-Ub (C), and ubiquitin C-terminally-fused DCNL1-UbCT (D) are shown. CUL5 neddylation was monitored by silver-stained SDS-PAGE and immunoblot analysis with anti-CUL5 antibody. Ratio of N8-CUL5 (in %) was determined from silver-stained SDS-PAGE using ImageJ software. Standard error of the mean is given from at least two independent replications.