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Fig. 4 | BMC Plant Biology

Fig. 4

From: Modulation of plant growth in vivo and identification of kinase substrates using an analog-sensitive variant of CYCLIN-DEPENDENT KINASE A;1

Fig. 4

Identification of CDKA;1 substrates by 2D-DIGE. a Strategy for identifying the kinase substrate by 2D-DIGE as applied here. For details see descriptions in the text. b In vitro kinase assay using wild-type and the analog-sensitive CDKA;1 (CDKA;1F80G) kinases together with CYCD2;1 as a cyclin partner using GST-RBR1-His6 as a substrate. After the kinase reaction with N6-PhEt-ATP-γ-S as a phospho-donor, proteins were alkylated with PNBM and were subjected to SDS-PAGE and transferred to a membrane. Thiophosphorylated RBR1 was detected with anti-thiophosphate ester antibody (top) and protein blot with anti-GST antibody (bottom) is showing an equal loading of the substrate. Mock was treated with 5 %(v/v) DMSO, the solvent of PNBM. Abbreviations: PNBM, p-nitrobenzyl mesylate, p-RBR1 for thiophosphorylated RBR1 resulting from kinase assays with N6-PhEt-ATP-γ-S. c A representative 2D-DIGE analysis. Protein extracts from wild-type seedlings incubated in the presence or absence of N6-PhEt-ATP-γ-S were labeled separately with Cy3 (532 nm, red) and Cy5 (635 nm, green), and proteins were then separated in the same gel in two dimensions and visualized by laser scanning. Most of the proteins from each treatment were focused similarly indicating a very low background level using N6-PhEt-ATP-γ-S. d A representative 2D-DIGE analysis. Protein extracts from cdka;1-as inflorescences incubated in the presence or absence of N6-PhEt-ATP-γ-S were labeled separately with Cy3 (532 nm, red) and Cy5 (635 nm, green). Proteins were separated and analyzed as in c. e Magnified image of C, showing that some spots were focused differently (arrow heads)

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