Skip to main content
. 2008 Nov 28;283(48):33211–33220. doi: 10.1074/jbc.M804440200

FIGURE 5.

FIGURE 5.

SCCRO does not overcome the inhibitory effects of CAND1 on neddylation. A, Western blot on GST-SCCRO pulldown products after incubation with purified, bacterially expressed Cul1-ROC1 and CAND1 showing CAND1 is pulled down by SCCRO only when it is complexed with Cul1-ROC1 (lanes 1 and 3). A CAND1 deletion (CAND1Δ604–1230) that loses binding to cullins is not pulled down by SCCRO (lane 4 and 5). B, Western blot from reciprocal GST-CAND1 pulldown assay showing interaction of CAND1 with SCCRO requires Cul1-ROC1 (lanes 2–4). C, Western blot for Cul1 following in vitro neddylation reaction using purified recombinant components showing complete inhibition of neddylation in the presence of 2-fold or higher molar excess of CAND1. D, Western blot on products from an in vitro neddylation reaction confirming increasing Cul1 neddylation with increasing amounts of SCCRO (lanes 2–4). The addition of CAND1 inhibits Cul1 neddylation (lane 5), which is not rescued even by 5-fold molar excess of SCCRO (lane 6; same blot, excess lanes removed). E, Western blot showing GST-CAND1 pulldown products after incubation with purified, bacterially expressed Cul1-ROC1, Ubc12∼NEDD8, and SCCRO. CAND1 interacts with Ubc12∼NEDD8 only when Cul1-ROC1 is present (lanes 1 and 2), and this interaction is enhanced by the presence of SCCRO (lane 3). F, Western blot on lysates from HA-SCCRO-transfected HeLa cells subjected to neddylation reaction showing an increase in cullin neddylation with the addition of ATP (lanes 1 and 2). HA immunoprecipitation (IP) of the same reaction products showed SCCRO preferentially interacts with unneddylated cullins (lanes 3 and 4). G, Western blot on products from an in vitro neddylation reaction using purified recombinant components showing an increase in the fraction of neddylated Cul1 with increasing reaction time (top panel). Western blot on the same reaction after GST-SCCRO (middle panel) and GST-CAND1 (bottom panel) pulldown assays showing that SCCRO and CAND1 interact with both free and neddylated cullins (same blot, excess lanes removed). H, Western blot on products from pulldown assays after GST-CAND1 complexed with either NEDD8-Cul1-ROC1 or free Cul1-ROC1 was incubated with increasing amounts of testis lysate from SCCRO+/+ mice showing release of NEDD8-Cul1-ROC1 but not unneddylated Cul1-ROC1. I, Western blot on products from pulldown assays after GST-CAND1 complexed with either NEDD8-Cul1-ROC1 or free Cul1-ROC1 was incubated with increasing amounts of testis lysate from SCCRO–/– mice showing release of Cul1-ROC1-NEDD8 only with addition of SCCRO and not SCCRO-D241N (same blot, excess lanes removed).