Fig. 1. Molecular characterization of the dE2F1^su89 mutation. (A) The point mutation (underlined) in the dE2F1^su89 C-terminus is shown. Amino acids that are conserved between Drosophila and mammalian E2Fs are shaded in gray, and the Rb-binding domain of dE2F1 is indicated with a black bar. (B) dE2F1^su89 did not interact with RBF in a yeast two-hybrid interaction assay. β-Gal activity on patches of yeast transformed with control plasmids or plasmids encoding RBF, wild-type dE2F1 or dE2F1^su89 (su89) is shown as indicated. (C) Endogenous dE2F1^su89 protein did not form a complex with RBF. Extracts from wild-type (WT) or dE2F1^su89 (su89) embryos were immunoprecipitated with anti-dE2F1, dDP and RBF antibodies as indicated, followed by western blot with an antibody against RBF or dE2F1 as indicated to determine the association between the endogenous RBF and dE2F1^su89 proteins. (D) dE2F1^su89 can activate transcription normally but is defective in its regulation by RBF. SL2 cells were transfected with no dE2F1 (1), a constant amount of wild-type dE2F1 (2–6) or dE2F1^su89 (7–11), and increasing amounts of RBF as indicated. The averages of normalized CAT activities from two independent experiments are shown.