FIGURE 3

dAPP-BP1 mutation blocks NEDD8 conjugation pathway. Accumulation of Ci^FL, Armadillo, and Cul1 in dAPP-BP1 mutant cells. (a–c) Clones carrying dAPP-BP1 ^Ex183 are marked by the lack of GFP expression (a, c). (a–i) The blue arrowheads mark the position of the morphogenetic furrow (MF) in this and following figures. For imaginal discs, anterior is to the left. (d–l) Clones carrying dAPP-BP1 ^Ex62 are marked by the lack of GFP (d, g) or beta-galactosidase (j, m) expression. (d–f) A late third-instar eye disc containing dAPP-BP1 ^Ex62 mutant clones and stained by anti-Ci^FL antibody. In mutant cells, Ci^FL protein levels (arrows) are similar in the MF and in the cells anterior to it. In posterior cells, Ci^FL persists, albeit at a lower level (arrowheads), and is not detected in the most posterior cells (asterisks). This pattern is similar to those observed in NEDD8 null clones.¹³ (g–i) A late third-instar eye disc containing dAPP-BP1 ^Ex62 mutant clones and stained with anti-Cul1 antibodies (red). Cul1 accumulates in dAPP-BP1 ^Ex62 mutant clones (arrows) that are marked by a lack of GFP expression (green in g, i). (j–l) Accumulation of Armadillo observed in dAPP-BP1 ^Ex62 mutant clones in the wing disc (arrows). (m–o) Accumulation of Ci^FL observed in the dAPP-BP1 ^Ex62 mutant clones in wig disc (arrows). (p) Ex62 is dAPP-BP1 ^Ex62 homozygote cell extracts. Western blot analysis of cell extracts from third-instar eye discs and brain lobes by anti-Cul1 antibodies indicates that the Cul1 protein is accumulated and the neddylated form of Cul1 (arrow) is almost missing in the dAPP-BP1 ^Ex62 homozygote