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. 2013 Mar 18;200(6):743–756. doi: 10.1083/jcb.201206065

Figure 1.

Figure 1.

Reduced CUL4B expression perturbs new origin firing. (A) Asynchronous cells were harvested and cytoplasmic (CF), nuclear-soluble (NF), and chromatin-bound (CBF) proteins were obtained and analyzed by Western blot. (B) Schematic double-labeled replication tracks and example of labeled replication tracks. Bar, 10 µm. (C) HeLa cells were transfected with CUL4B (siCUL4B) or negative control (siCon) siRNA. 72 h after transfection, levels of total cellular CUL4A and CUL4B were determined by Western blot analysis. (D) HeLa cells transfected with siCUL4B or siCon siRNA were synchronized in G1/S phase by double-thymidine block. 3 h after release into S phase, cells were labeled sequentially with CldU and IdU. At least 200 CldU/IdU-positive DNA fibers were visualized and quantified using a fluorescence microscope; results represent means ± SD from three independent experiments. (E) Indicated HeLa cells were synchronized with the double-thymidine block and released to progress through the cell cycle. Cells were harvested at the indicated times for analysis of cell cycle distribution by flow cytometry. The data shown are from a representative of three independent experiments.