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Figure 3 | Plant Methods

Figure 3

From: Robust reconstitution of active cell-cycle control complexes from co-expressed proteins in bacteria

Figure 3

Activity of SDS containing CDK-cyclin complexes. For both panels, the first and third rows show kinase assays using bovine Histone H1 as a generic substrates as well as RBR1 from Arabidopsis that was expressed and purified from bacteria. Second and forth panels depict the results of CBB staining of the same SDS-PAGE gel indicating equal amount of substrates in each reaction. The fifth row (CDKs) shows western blot analyses of the respective pull-downs using Strep-Tactin HRP (either CDKA;1, CDKB1;1, or CDKB2;2 as indicted in the header of each panel) demonstrating that the respective CDKs bound to the particular cyclin that was co-expressed. (a) Complexes binding to a Ni-NTA column (His-tagged proteins) were purified from bacteria either expressing HisMBP-tagged putative cyclin SDS together with Cak1, or HisMBP-SDS together with Cak1 and StrepIII-tagged CDKA;1, or HisMBP-SDS together with Cak1 and StrepIII-tagged CDKB1;1, or HisMBP-SDS together with Cak1 and StrepIII-tagged CDKB2;2. Only CDKA;1-SDS but not CDKB1;1-SDS and CDKB2;2-SDS complexes isolated from E. coli extracts have high activity against both the generic substrate Histone H1 and Arabidopsis RBR1. (b) Complexes binding to a Ni-NTA column (His-tagged proteins) were purified from cells either expressing HisMBP-tagged cyclin CYCB1;2 together with Cak1, or HisMBP-CYCB1;2 together with Cak1 and StrepIII-tagged CDKA;1, or HisMBP-CYCB1;2 together with Cak1 and StrepIII-tagged CDKB1;1, or HisMBP-CYCB1;2 together with Cak1 and StrepIII-tagged CDKB2;2. CDKB1;1-CYCB1;2 and especially CDKB2;2-CYCB1;2 but only weakly CDKA;1-CYCB1;2 complexes isolated from E. coli extracts have activity against the two substrates Histone H1 and RBR1. Note that kinase assays between different panels, e.g. (a) and (b) cannot a priori be compared due to different levels of precipitated kinase complexes and the exposure time. Abbreviations: CBB Coomassie brilliant blue. p-Histone H1 for radio-labeled Histone H1 resulting from kinase assays with radio-labeled ATP.

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