Fig. 5. Effect of the NEDD8 system on recruitment of E2 (Ubc4) to the ROC1–SCF^βTrCP1 complex. (A) Effect of Ubc4 binding to ROC1–SCF^βTrCP1 and ROC1–SC(K/R)F^βTrCP1. Experiments and symbols are similar to those described in Figure 4, except that IκBα and IKK were not added and immunoprecipitation was carried out with anti-Cul-1 antibody. Some experiments were conducted in duplicate. (B) Only Ub-linked Ubc4 was recruited onto ROC1–SCF^βTrCP1. The experiment was similar to that described in (A) except that E1 (Uba1) was omitted from the assay mixture. (C) The NEDD8 system does not support the binding of Ubc4(C/A) and Ubc4(C/S) to ROC1–SCF^βTrCP1. The experiment was similar to that described in (A), except that Ubc4(C/A) and Ubc4(C/S) were used for wild-type Ubc4 (wt). (D) The NEDD8 system does not support the binding of Ubc4(C/A) and Ubc4(C/S) to pIκBα. The experiment was similar to that described in Figure 5 using Ubc4(C/A) and Ubc4(C/S). (E) Ubc4, but not Ubc4(C/A), co-immunoprecipitates Cul-1 in 293 cells. Thirty-six hours after pcDNA3-Cul-1-HA with unmodified FLAG-Ubc4(wt) or FLAG- Ubc4(C/A) were transfected into 293 cells, crude extracts were prepared as described in Materials and methods. After immuno precipitation by anti-FLAG antibody, the resulting immunoprecipitates were analysed by western blotting using anti-HA antibodies.