FIG. 5.

(A) The decay rate of DDB2 is specifically enhanced by the expression of Cul-4A. Five plates of HeLa cells were transfected with plasmids that express T7 epitope-tagged DDB2 alone or with plasmids expressing V5-tagged Cul-4A or V5-Cul-1 or V5-Cul-4A (dl 1–52) or Cul-4B. At 16 h posttransfection, the cells from same set were trypsinized, pooled, and replated on five plates. After 24 h, the cells were treated with cycloheximide (20 μg/ml) and harvested at the indicated time points. Then 100-μg portions of the transfected cell extracts were analyzed by Western blot assays. The blot was probed with antibodies against the T7 epitope. (B) Plot showing the decay rate of DDB2 in the presence of different cullins. The band intensities were determined by densitometric scanning. For each plot, the band intensity corresponding to the zero hour time point was taken as 100% and the relative intensities of the bands at various time points were plotted. Symbols: ⧫, DDB2; ■, DDB2 + Cul-1; ▵, DDB2 + Cul4A; ×, DDB2 + Cul-4B; ∗, DDB2 + Cul-4A (dl 1–52).