Xenbase Image ID: 131503

Figure 3. The Lag Time between the Cdc25 Shift and the Cdc27 or MP44 Hypershifts Is a Function of the Substrates and Not the Presence of Regulators in the Extract Both Cdc27 ([A], top panel) and MP44 (B) are phosphorylated in steps and require more time in mitotic extracts to become fully phosphorylated than Cdc25 ([A], bottom panel), which shifts abruptly at the G2/M transition. (A) In vitro-translated Cdc27 labeled with [35S]methionine was added to Xenopus interphase egg extracts at room temperature for 40 min to allow exchange of IVT protein into the APC. The extract plus Cdc27 and IVT 35S-labeled Cdc25 were added simultaneously to extracts at 10, 15, 20, or 30 min post cyclin B addition, and samples were taken for gel analysis. Control reactions contained IVT Cdc27 and Cdc25 added to interphase extracts at the same time as cyclin B (0 min). The top set of labels above the panel, Age of the Extract, refers to the length of time between addition to the extracts of GST-cyclin B and substrate. Time points immediately above the panels indicate the cumulative time since addition of GST-cyclin B to the extracts, and those below the panels indicate the length of time since Cdc25 and Cdc27 were added to the extracts. In the latter case, the 0 min time point is set to the Cdc25 shift as an internal reference point. (B) IVT MP44 protein was added to interphase extracts at the same time as GST-cyclin B or at 20 or 40 min after cyclin B addition. The top set of labels represents the Age of the Extract as described above. The labeling of the time points above the panels is the total time after Gst-cyclin B addition as above, while the time points below the panel indicate the elapsed time from substrate (MP44) addition. (C) Graph of three domains of the MP44 shift. The intensity of the signal for each time point was calculated by integrating of the values determined by densitometry tracings of the gels for each of the shifts (see Experimental Procedures). Image published in: Georgi AB et al. (2002) Copyright © 2002. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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