Interaction of the Retinoblastoma Protein with Orc1 and Its Recruitment to Human Origins of DNA Replication
Figure 6
Cell cycle-dependent association of Rb and E2F1 with the Lamin B2 origin.
(A) Experimental scheme for HeLa cell synchronization. HeLa cells were synchronized in mitosis by a double thymidine/nocodazole block, and then followed G1 after release from the block. (B) Flow cytometry profiles of asynchronous cells (Asynch), cells blocked in mitosis (0 hr) or cells at different times after release. (C) Western blot analysis of whole-cell extracts obtained from cells at different time points during synchronization. (D) Quantification of cross-linked lamin B2 origin DNA immunoprecipitated by ChIP. On top of the graph, the antibodies used for ChIP are shown. The histogram reports the results (mean±sem) of at least three independent experiments. The results are presented as the fold enrichment of the lamin B2 origin region (B48) over the irrelevant B10 region, after normalization for the levels of immunoprecipitated chromatin using an unrelated antibody (normal rabbit IgG) as control. (E) Experimental scheme for T98G cell synchronization. Cells were cultured without serum for 72 h and then followed for 20 h after addition of serum. (F) Flow cytometry profiles of asynchronous cells (Asynch), cells blocked in G0 by serum starvation (0 hr) or cells at different times after serum stimulation. (G) Western blot analysis of whole-cell extracts obtained from cells at different times points during synchronization. (H) Results of ChIP experiments for the lamin B2 origin, using the antibodies indicated on top of each dataset. The results are presented as in (D). AcH3: acetylated histone H3; AcH4: acetylated histone H4.
