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Fig. 8. Clb1 and Clb2 proteins carry functional destruction boxes. (A) Scheme showing the Clb1 and Clb2 proteins, as well the different deletion mutants. The sequences for the destruction boxes are indicated. These constructs were expressed under the control of a less-active version of the Pcrg1 promoter to avoid the problems associated to high cyclin levels (see text for details). (B) Expression levels of Clb1 and Clb2 derivates. Cells were grown in non-induction (complete medium with 2% glucose, CMD) and induction (complete medium with 2% arabinose, CMA) conditions for 6 hours. Whole cell lysates were separated by SDS-PAGE and immunoblotted with anti-VSV (Clb1 western, upper panel) or anti-MYC (Clb2 western, lower panel) antibodies. Similar amount of total extract was loaded per lane. Upper panel: TAU36-2 (Clb1), TAU30 (Clb1{Delta}db1), TAU45 (Clb1{Delta}db2) and TAU31 (Clb1{Delta}db1-2) cells that express VSV-tagged versions of Clb1 and derived proteins. The different level of protein expression could be attributed to a lower translation efficiency in the derivates lacking the N-terminal destruction box. Lower panel: TAU52-2 (Clb2) and TAU53 (Clb2{Delta}db) cells that express MYC-tagged versions of Clb2 and Clb2{Delta}db proteins. (C) Serial tenfold dilutions of exponential cultures of FB1 (control), TAU36-2 (Pcrg*-Clb1), TAU30 (Pcrg*-Clb1{Delta}db1), TAU45 (Pcrg*-Clb1{Delta}db2), TAU31 (Pcrg*-Clb1{Delta}db1-2), TAU52-2 (Pcrg*-Clb2), and TAU53 (Pcrg*-Clb2{Delta}db) strains were spotted on complete medium plates with glucose (CMD) or arabinose (CMA) as sole carbon source. Plates were incubated at 28°C for 3 days. (D) Deletion of D-boxes stabilizes Clb1 and Clb2. TAU36-2, TAU31, TAU52-2 and TAU53 cells were grown for 1 hour in complete medium with 2% arabinose, and then transferred to completed medium with 2% glucose and 100 µg/ml cycloheximide. Samples were taken at the indicated time, and Clb1, Clb2 and Cdk1 levels were analysed by immunoblotting with anti-VSV, anti-MYC and anti-PSTAIRE antibodies respectively.





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