Figure 3.

CUL4B promotes CDC6 loading onto chromatin. (A) Indicated cells were synchronized at the G1/S boundary and cytoplasmic (CF), nuclear-soluble (NF), and chromatin-bound proteins (CBF) were prepared. Equal amounts of protein extracts were subjected to SDS-PAGE and detected with the indicated antibodies. (B) Western blot analysis of CUL4B, CDC6, and MCM2 protein levels in the chromatin-bound fraction prepared from asynchronous cells indicated. (C) miCUL4B and control HeLa cells were transfected with indicated plasmids. 72 h after transfection, chromatin-bound proteins were extracted and analyzed by Western blot. (D) Cells were transfected with indicated plasmids. At 72 h after transfection, chromatin-bound proteins were extracted and analyzed by Western blot. (E) HeLa cells cotransfected with indicated siRNAs and plasmids were synchronized in G1/S phase by double-thymidine block. 3 h after released into S phase, cells were labeled sequentially with CldU and IdU. DNA fibers were immunostained with anti-CldU and anti-IdU antibodies. More than 200 CldU/IdU-positive DNA fibers were visualized and quantified using a fluorescence microscope; results represent means ± SD from three independent experiments.