Fig. 3.

DDB1-CUL4A^DDB2 E3 fails to bind to chromatin after UV irradiation of XP-E cells. The solubilized chromatin fractions were immunoprecipitated with rabbit DDB2- and DDB1-IgY antibodies. The fractions (Input) were obtained after MNase complete digestion of the chromatin-bound fractions prepared from 8 × 10⁶ normal and XP-E lymphoblastoid cells that had not been irradiated or had been allowed to recover for 45 min after irradiation (30 J/m²). The coimmunoprecipitates were analyzed by immunoblotting with DDB1, CUL4A, and DDB2sc antibodies to the components of DDB1-CUL4A^DDB2 ubiquitin ligase. The cytosolic, 0.3 M nuclear soluble, and solubilized chromatin fractions (see Materials and Methods) were also included in the Western blot to illustrate the difference between the subcellular distribution of mutated and normal DDB2.