UbcD4, a ubiquitin-conjugating enzyme in Drosophila melanogaster expressed in pole cells

Canning, Mary; Kirby, Ruth; Finnegan, David J.

doi:10.1007/s00438-001-0623-8

Cited by 1 publication

(1 citation statement)

References 30 publications

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“…We used HRP-linked anti-biotin antibody from Cell Signaling Technology at 1:100, rabbit polyclonal anti-Ub from Sigma at 1:250, FK1 anti-poly-Ub from EnzoLifeSciences at 1:1000, rabbit polyclonal anti-Ube1 (to detect its Drosophila ortholog Uba1) from Abcam at 1:100, sheep polyclonal anti-UbcD4 (21) at 1:100, rabbit polyclonal anti-E2-14K (to detect its Drosophila ortholog Ubc-E2H) from Boston Biochem at 1:300, rabbit polyclonal anti-UbcH6 (to detect its ortholog UbcD2) from Boston Biochem at 1:300, mouse polyclonal anti-Eff (22) at 1:1000, rabbit polyclonal anti-UBE2G1 (to detect its ortholog CG40045) from Boston Biochem at 1:300, mouse monoclonal anti-neurotactin (Nrt) from the Developmental Studies Hybridoma Bank (DSHB) at 1:20, mouse monoclonal anti-Fas2 from DSHB at 1:25, rabbit polyclonal anti-Eps15 (23) at 1:250, rabbit polyclonal anti-fax from Eric Liebl at 1:1000, rabbit polyclonal anti-Rad23 (24) at 1:1000, mouse monoclonal anti-Pros54 (24) at 1:2, rabbit polyclonal anti-ArgK (25) at 1:100, mouse monoclonal anti-Hsp27 (26) at 1:20, mouse monoclonal anti-Syx1A from DSHB at 1:50, guinea pig polyclonal anti-Lqf (27) at 1:200, rabbit polyclonal anti-anaplastic lymphoma kinase (28) at 1:200, and mouse monoclonal anti-FLAG M2 (Sigma) at 1:1000. HRP-conjugated secondary antibodies from Jackson ImmunoResearch Laboratories were used at dilutions between 1:2000 and 1:5000.…”

Section: Methodsmentioning

confidence: 99%

A Novel Strategy to Isolate Ubiquitin Conjugates Reveals Wide Role for Ubiquitination during Neural Development

Franco

Seyfried²,

Brand

et al. 2011

Molecular & Cellular Proteomics

106

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Ubiquitination has essential roles in neuronal development and function. Ubiquitin proteomics studies on yeast and HeLa cells have proven very informative, but there still is a gap regarding neuronal tissue-specific ubiquitination. In an organism context, direct evidence for the ubiquitination of neuronal proteins is even scarcer. Here, we report a novel proteomics strategy based on the in vivo biotinylation of ubiquitin to isolate ubiquitin conjugates from the neurons of Drosophila melanogaster embryos. We confidently identified 48 neuronal ubiquitin substrates, none of which was yet known to be ubiquitinated. Earlier proteomics and biochemical studies in non-neuronal cell types had identified orthologs to some of those but not to others. The identification here of novel ubiquitin substrates, those with no known ubiquitinated ortholog, suggests that proteomics studies must be performed on neuronal cells to identify ubiquitination pathways not shared by other cell types. Importantly, several of those newly found neuronal ubiquitin substrates are key players in synaptogenesis. Mass spectrometry results were validated by Western blotting to confirm that those proteins are indeed ubiquitinated in the Drosophila embryonic nervous system and to elucidate whether they are mono- or polyubiquitinated. In addition to the ubiquitin substrates, we also identified the ubiquitin carriers that are active during synaptogenesis. Identifying endogenously ubiquitinated proteins in specific cell types, at specific developmental stages, and within the context of a living organism will allow understanding how the tissue-specific function of those proteins is regulated by the ubiquitin system.

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