Fig. 1. (A) The putative cdk phosphorylation sites in RBF and the different RBF mutants generated. There are two other potential phosphorylation sites (T83 at the N terminus and T610 in the middle of the pocket domain) in RBF that are not mutated and are not shown. (B) The effects of Cyclin D/Cdk4 and Cyclin E/Cdc2c on wild-type RBF and RBF phosphorylation site mutants. SL2 cells were transfected with the E2F4CAT reporter construct, together with the E2F1/DP, Cyclin D/Cdk4, Cyclin E/Cdc2c, and the different RBF constructs as indicated. Basal indicates cells transfected with E2F4CAT reporter only. D/Cdk4 represents Drosophila Cyclin D and Cdk4. E/Cdc2c represents Drosophila Cyclin E and Cdc2c. ‘+’ indicates that a given construct was co-transfected; ‘-’ indicates that the construct was not co-transfected. (C) A yeast two-hybrid interaction assay to test the interaction between several RBF mutants and dE2F1. Various patches of yeast contained the pPC86-dE2F1 plasmid and the plasmids as indicated: Control, pPC97; WT RBF, pPC97-RBF(WT); RBF-30, pPC97-RBF-30; RBF-596, pPC97-RBF-596W; RBF-555, pPC97-RBF-555L. Patches of cells growing on plates selective for the presence of both plasmids were tested for the ß-galactosidase activity.