FIG. 4.

Coexpression of Cul-4A specifically reduces the half-life of DDB2. Five plates (10-cm-diameter dish) of HeLa cells were transfected with plasmids that express T7 epitope-tagged DDB1 and DDB2 in the presence or absence of a plasmid expressing V5-tagged Cul-4A. At 16 h posttransfection, the cells were trypsinized and the cells from the same set were pooled and replated on five plates. After 24 h, cycloheximide was added to the medium at a final concentration of 20 μg/ml and the cells were harvested at the indicated time points. Total-cell extracts were prepared as described in Materials and Methods. 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.