Interaction and colocalization of E2FBP1 and ICP0 in HEK293FT and TIG-3 cells. (A) Interaction between E2FBP1 and ICP0 was detected by immunoprecipitation of cell lysates. HEK293FT cells were transformed with pDS16 (ICP0), pcDNA3-2HA-E2FBP1 (HA-E2FBP1), and pcDNA3 (empty vector) and cultivated for 18 h. Cell were treated with 10 μM (each) proteasome inhibitors MG115 and MG132 for 15 min, and lysates were prepared and subjected to immunoblotting and immunoprecipitation. The numbers on top of the lanes indicate the relative amounts of the plasmids. Lanes 1 to 3 received 20 μg of cell lysate, whereas lanes 4 to 6 contained coprecipitated materials obtained from 2 mg of cell lysates incubated with either anti-ICP0 (5H7) or anti-HA (3F10) antibody. WCL, whole-cell lysate; IB, immunoblotting; IP, immunoprecipitation. (B) Endogenous E2FBP1 and ICP0 expressed from transformed plasmid DNA colocalized in PML-NB-like nuclear subdomains. TIG-3 cells treated with or without E2FBP1 siRNA (siE2FBP1) or nonsense control siRNA (siControl) were subsequently transformed with pDS16. After 2 days, cells were stained with anti-ICP0 (5H7) (green), anti-DRIL1 (red), and DAPI (gray), and images were captured by confocal microscopy.