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Figure 4


Fig. 4. Degradation profile of the Clb2{Delta}NLS mutant protein. (A) Comparison of Clb2 levels in metaphase and telophase-arrested cells. WT and cdc15-2 cells expressing the indicated CLB2 alleles were exponentially grown at 25°C and transferred at 37°C with (WT background, metaphase arrest) or without (cdc15-2 background, telophase arrest) 15 µg/ml nocodazole for 2 hours. Metaphase arrests were performed at 37°C to normalize the temperature shift required to block cells in telophase. Cellular extracts were analyzed by western blot with the indicated antibodies. Signals from Alexa Fluor-coupled secondary antibodies were quantified with an Odyssey scan. Clb2 signals were normalized using Cdc28 as a loading control. Signals from metaphase-arrested cells were considered as 100%. (B) Kinetics of Clb2 and Clb2{Delta}NLS degradation during mitotic exit. cdc15-2/cdc15-2 CLB2/clb2{Delta}NLS diploid cells were released from a temperature shift-induced telophase arrest as described in Materials and Methods. Clb2 signals were normalized using Cdc28 as a loading control and are expressed as the percentage of the signals measured at t0 (100%).





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