Fig. 5.

CDK phosphorylates MYB3R3 and MYB3R5. a In vitro kinase assay of His-MYB3R3 and His-MYB3R5. Protein extracts from Arabidopsis MM2d cultured cells were immunoprecipitated with specific antibodies against CDKA, CDKB1 and CDKB2, and assayed for kinase activity using His-MYB3R3 or His-MYB3R5 as substrate. His-GFP was used as a control. Relative intensity of each band is expressed as the fold change, normalized with respect to the band of substrates visualized by CBB staining. b Schematic representation of MYB3R3 harbouring three Myb domains. Arrowheads indicate CDK phosphorylation sites in the C-terminal region. Black bars indicate protein fragments used for kinase assay in c. c Kinase assay of partial fragments of MYB3R3. Protein extracts from MM2d cells were immunoprecipitated with the anti-CDKA antibody and assayed for kinase activity using His-tagged fragments of MYB3R3 (F1, F2 and F3) as substrate. Arrowheads indicate the position of each substrate. d Kinase assay of His-MYB3R3 with alanine substitutions. Protein extracts from MM2d cells were immunoprecipitated with the anti-CDKA antibody and assayed for kinase activity using His-MYB3R3 with alanine substitutions at CDK phosphorylation sites as substrate. Relative intensity of each band is expressed as the fold change, normalized with respect to the band of substrates visualized by CBB staining