FIG. 8.

DDB2 is a specific ubiquitination target of Cul-4A. (A) Cells were transfected with plasmids expressing T7-DDB2 or His-tagged ubiquitin in combination with empty vector, V5-Cul-1, V5-Cul-4A, V5-Cul-4A(dl 1–52), or V5-Cul-4B. At 40 h after transfection, the cells were treated with MG 132 for 5 h. Total-cell extracts were prepared as described in Materials and Methods. Equal amount of extracts were treated with 2% SDS at 37°C for 10 min. The SDS-treated extracts were diluted 10-fold and then subjected to immunoprecipitation (IP) with T7 antibodies and Western blot assay with monoclonal antibodies against ubiquitin (Ub-ab). (B) Level of expression of T7-DDB2 in the extracts. Transfected cell extracts (100 μg) were probed with T7- antibody in a Western blot to assay for DDB2 expression.