Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2011 Sep 8;6(9):e24168. doi: 10.1371/journal.pone.0024168

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

Du et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

PMC Copyright notice

(A) Immunoblot analysis (WB) of lysates extracted from wildtype (WT) wing discs (lane 1) or wing discs overexpressing CG7375 RNAi driven by the MS1096-Gal4 driver (lane 2). CG7375 RNAi led to accumulation of both CiFL and un-neddylated Cul1 (lane 2). Similarly, neddylation of Cul3 was reduced, but the amount of un-neddylated Cul3 was not obviously changed (lane 2). (B) Immunoblot analysis of lysates extracted from wildtype (lane 1) or homozygous CG7375^LL04684 first-instar larvae (lane 2). Neddylation of Cul3 protein was abolished and un-neddylated Cul3 was stabilized. (C and D) The E2 activity of CG7375 for Cullin neddylation. In an in vitro neddylation assay, purified human Uba3/APPBP1 complex was used as E1 and lysates extracted from wing discs expressing CG7375 RNAi provided the source for Cullin proteins. CG7375 RNAi wing disc lysates did not display neddylation activity (as 90% endogenous CG7375 was knocked down by CG7375 RNAi; Figure S5C), unless purified GST-CG7375 protein was added (lane 2 in C; lane 3 in D): both Cu1 and Cul3 were neddylated. Purified GST protein was used as a negative control (lane 1 in C and D). The neddylation activity of GST-CG7375 was dependent on the presence of purified E1 complex (lane 3 in C) and ATP (lane 4 in C). The N-terminus of human ortholog of CG7375 (Ubc12) is required to selectively recruit NEDD8's E1 to promote thioester formation between E2 and NEDD8 (Figure S7). Deletion of this conserved N terminal motif in GST-CG7375DN abolished its neddylation E2 activity (lane 2 in D). (E) In vitro reconstitution of Drosophila neddylation cascade. Cul1 and Cul3 were neddylated when both E1 complex (CG13343-V5 and dAPPBP1-HA produced in cl-8 cells) and E2 enzyme (GST-CG7375) were added to cl-8 lysates, which provided the source of Cullins (lane 4). Adding E1 (lane 2) or E2 (lane 3) alone did not result in neddylation of Cul1 or Cul3.