FIG. 6.

(A) Cul-4A induces ubiqitination of DDB2. HeLa cells were transfected with plasmids expressing T7-DDB2 (4 μg), T7-DDB1 (6 μg), T7-2RO (4 μg), His-tagged ubiquitin (U6) (1 μg), and V5-Cul-4A (9 μg) in the indicated combinations. At 40 h after transfection, the cells were treated with MG 132 for 5 h. The cells were washed and lysed as described in Materials and Methods. Equal amount of extracts (1 mg) were immunoprecipitated (IP) with T7 antibodies (ab) covalently linked to agarose beads, and the immunoprecipitates were subjected to Western blot assay. The blot was probed with monoclonal antibodies against ubiquitin. (B) Western blot of the extracts (100 μg) probed with T7 antibody (ab) to assay for the expression levels of DDB2 in the above extracts.