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Fig. 3. Neddylation of Cul1 and Cul3 in DEN1 mutants. (A,B) Deneddylation of Cul1 (A) and Cul3 (B) by DEN1. Neddylated and unneddylated Flag-Cul1 or Flag-Cul3 proteins were immunoprecipitated by the anti-Flag antibody from extracts of S2 cells transfected with pUAST-myc-mNedd8 and pUAST-flag-Cul1 or pUAST-flag-Cul13. The precipitates were reconstituted in reactions with purified DEN1 (0, 2.5, 5 µg in lanes 1, 2, 3, respectively) and DEN1CA (5 µg in lane 4). Western blots against Cul1 (A) or Cul3 (B) show that the protein levels of neddylated Cul1 and Cul3 are reduced in the presence of DEN1 but not DEN1CA. (C,D) Protein extracts prepared from wild-type and DEN1^null larvae were analyzed by western blots for Cul1 (C) and Cul3 (D) expression. The levels of neddylated Cul1 and Cul3, denoted as Cul1^Nedd8 and Cul3^Nedd8, are reduced in DEN1^null mutants. (E,F) UAS-myc-mNedd8 was expressed by tubP-GAL4 in wild-type and DEN1^null, and larval lysates were immunoprecipitated by antibodies against Cul1 (E) or Cul3 (F). The immunocomplexes were analyzed using anti-Cul1 (left in E), anti-Cul3 (left in F) and anti-Myc antibodies (right panels) in western blots. Cul1^Nedd8 levels are dramatically reduced, whereas the Cul3^Nedd8 levels are only slightly reduced.

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