Interaction of the Retinoblastoma Protein with Orc1 and Its Recruitment to Human Origins of DNA Replication
Figure 5
(A) HeLa cells were transiently transfected with expression vectors coding for the proteins indicated on top of each panel fused to either EGFP (green color) or BFP (blue color). Individual transfected cells were visualized by excitation at 480 nm and collection at 520 nm, showing EGFP fluorescence after direct EGFP excitation (panels in the upper row), and by excitation at 350 nm and collection at 520 nm, showing EGFP fluorescence after BFP excitation, indicating FRET (panels in the lower row). The box plot below each image pair shows the quantification of FRET. Fluorescent emission at 520 nm from individual cells was recorded after excitation at 350 or 480 nm, and integrated intensities over the whole cell were evaluated. The percentile box-plot distribution of the ratio between these two measurements is shown by considering at least 10 consecutively analyzed cells for each protein pair. Horizontal lines of the percentile box plot distribution, from top to bottom, mark the 10th, 25th, 50th, 75th, and 90th percentile respectively. (B) FRET between Rb, tagged with BFP, and the same set of Orc1 truncation mutants considered for the GST pulldown experiments, tagged with EGFP.
