| Texto completo | |
| Autor(es): |
dos Passos, Patricia M. S.
;
Spagnol, Valentine
;
de Correia, Camila R. S. T. B.
;
Teixeira, Felipe R.
Número total de Autores: 4
|
| Tipo de documento: | Artigo Científico |
| Fonte: | JOVE-JOURNAL OF VISUALIZED EXPERIMENTS; v. N/A, n. 183, p. 11-pg., 2022-05-01. |
| Resumo | |
Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, including cell survival, proliferation, and differentiation. It is a reversible process that consists of a covalent attachment of ubiquitin to the substrate through a cascade reaction of at least three different enzymes, composed of E1 (Ubiquitin-activation enzyme), E2 (Ubiquitinconjugating enzyme), and E3 (Ubiquitin-ligase enzyme). The E3 complex plays an important role in substrate recognition and ubiquitylation. Here, a protocol is described to evaluate substrate ubiquitylation in mammalian cells using transient co-transfection of a plasmid encoding the selected substrate, an E3 ubiquitin ligase, and a tagged ubiquitin. Before lysis, the transfected cells are treated with the proteasome inhibitor MG132 (carbobenzoxy-leu-leu-leucinal) to avoid substrate proteasomal degradation. Furthermore, the cell extract is submitted to small-scale immunoprecipitation (IP) to purify the polyubiquitylated substrate for subsequent detection by western blotting (WB) using specific antibodies for ubiquitin tag. Hence, a consistent and uncomplicated protocol for ubiquitylation assay in mammalian cells is described to assist scientists in addressing ubiquitylation of specific substrates and E3 ubiquitin ligases. (AU) | |
| Processo FAPESP: | 20/15771-6 - Caracterização funcional das E3 ubiquitina-ligases do tipo CRL (Cullin RING-ligases) em Leishmania infantum |
| Beneficiário: | Felipe Roberti Teixeira |
| Modalidade de apoio: | Auxílio à Pesquisa - Regular |
| Processo FAPESP: | 16/20258-0 - Leishmaniose visceral: abordagens genômicas para análise molecular integrada do hospedeiro e do parasito |
| Beneficiário: | Sandra Regina Costa Maruyama |
| Modalidade de apoio: | Auxílio à Pesquisa - Jovens Pesquisadores |