FIGURE 4. Relationship between Myc protein levels and the number of active DNA replication origins. a, Replication reactions in mock- and XMyc-depleted Xenopus cell-free extracts were stopped at the indicated time points and quantified. An average of three experiments is shown (plusminuss.d.). b, Electrophoretic analysis of nascent replication products on Ara-C-induced synchronization of depleted Xenopus extracts (top panel). Bottom panel: average size of replication products plotted over time to indicate elongation rates (best-fit curves). dCTP, deoxy-cytidine triphosphate. c, Replication reactions in Xenopus extracts, supplemented with increasing amounts of XMyc, were stopped at 60 min and analysed. d, Top panel: replication reactions in Xenopus extracts assembled in the presence or absence of XMyc and analysed after 40 min. Bottom panel: western blot analysis of corresponding chromatin fractions. Chromatin-bound Xenopus Cdc45 was quantified using Scion software and normalized to Xenopus MCM6. e, Analysis of BrdU foci unclear distribution in synchronized WI38 fibroblasts (Myc-ER or empty vector). alpha-Amanitin (2 mug ml-1) or buffer was added 30 min before tamoxifen (4-OHT) exposure and BrdU labelling (see scheme in Supplementary Fig. 10). f, Quantification of the experiment shown in e. Nuclear BrdU intensity is the sum of all BrdU spots. The threshold is set above 3 s.d. from background. Asterisk, P < 0.05, based on a two-sided Student's t-test. Each dot is the sum intensity of one cell. pB, empty vector; T, tamoxifen; A, alpha-amanitin. g, Transiently transfected H1299 cells (GFP with or without c-Myc cDNA), synchronized in G1/S and released to S phase in the presence of 32P orthophosphate. After 3 h, DNA was isolated and incorporated nucleotides quantified by scintillation counting. Bottom panel: western blot analysis of whole-cell lysates from the same cells. h, H1299 cells were co-transfected with an HA-tagged Cdc45 cDNA and empty vector or c-Myc cDNA. On synchronization, cells were collected in S phase and chromatin fractions (CHR) or whole-cell extracts (WCE) resolved by SDS–PAGE and analysed by western blot.
Image published in: Dominguez-Sola D et al. (2007)
Copyright © 2007. Image reproduced with permission of the Publisher, Macmillan Publishers Ltd.
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