Fig. 1.

DDB1-CUL4A^DDB2 ubiquitin ligase colocalizes with UV-damaged DNA in vivo. (A) Transformed human fibroblasts (WI-38VA13 cells) were transiently transfected with full length CUL4A- or DDB2-expressing cDNAs. Forty hours after transfection, the cells were irradiated through an 8-μm pore filter with a dose of 60 J/m². Immediately after treatment, the cells were washed with CSK buffer (100 mM NaCl/300 mM sucrose/10 mM Pipes, pH 7.0/3 mM MgCl₂, and protease inhibitors), incubated for 5 min in CSK plus 0.2%Triton X-100, and then fixed. Cells were counterstained with DAPI (blue). UV-irradiated sites were visualized by fluorescent immunostaining by using anti-CPD antibody (red). CUL4A was visualized by an antibody to the V5 epitope present on the transfected Cul4A (green). DDB2 was visualized by an antibody to the Flag epitope present on the transfected DDB2 (green), and endogenous DDB1 was visualized by a DDB1-IgY antibody (green). Merge shows the protein colocalization with damaged DNA. (B) The experiment was performed as described in A, except that the plasmids with CUL4A and DDB2 were cotransfected into WI-38VA13 cells. Merge shows that V5-CUL4A and Flag-DDB2 colocalize in the UV-irradiated subnuclear spot.