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. 2015 Jan 20;14(7):1010–1023. doi: 10.1080/15384101.2015.1007003

Figure 2.

Figure 2.

Assembly of GAL4 pre-RC on the plasmids proximal to GAL4 DNA-binding sites. (A) Schematic diagram of the 7.6 kbp plasmid. The location of 4 primer sets (X, Y, Y′, and Z) used for the ChIP analyses, and 6× GAL4 DNA-binding sites (light green) are indicated. (B) ChIP analysis of pre-RC components bound to the plasmids. The bead-coupled plasmids with (+) and without (-) the DNA-binding sites were incubated with GAL4-Cdc6, and the pre-RC was assembled as described in Figure 1. The localization of pre-RC components was examined by ChIP. Antibodies used for immunoprecipitation are indicated at the top of each panel. The amount of target DNA in each IP sample was determined by qPCR, and relative enrichment was estimated by dividing the amount obtained with each IP sample by that of the mock IP sample using control antibody. Average data are shown as a bar graph with standard deviation (SD) indicated by bars from 3 independent PCR reactions of each ChIP sample. (C) Effect of geminin on the assembly of GAL4 pre-RC on the plasmids. The bead-coupled plasmids with the DNA-binding sites were pre-incubated with GAL4-GST (GST) or GAL4-Cdc6 (Cdc6), and the assembly of pre-RC components in the presence (+) and absence (−) of GST-geminin (1 μM) was analyzed as in (B).