FIGURE 10.

Cross-linking between K679C Cul1-Rbx1 and DPS-activated Cdc34 proteins is affected by the ionic strength of the reaction buffer and is dependent on the presence of the Cdc34 acidic tail. A, Cul1 Western blot of reactions between 227C Cdc34 2C and K679C Cul1-Rbx1 with increasing concentrations of NaCl in the presence of DPS. Note that K679C Cul1 was chosen because Lys-679 is centrally located in the basic canyon region. B, graph showing the fraction of K679C Cul1 that was converted into Cdc34-Cul1 products as a function of the concentration of NaCl in the reaction buffer. C, cross-linking between K679C Cul1-Rbx1 and either S36C Δ190 or K167C Δ190 Cdc34 2C proteins. DPS-activated Cdc34 proteins were incubated with K679C Cul1-Rbx1 for the indicated amounts of time, followed by SDS-PAGE and detection of proteins by Cul1 Western blotting.