35 research outputs found
Autophagy mediates degradation of nuclear lamina
Z.D. is supported by a fellow award from the Leukemia & Lymphoma Society. B.C.C. is supported by career development awards from the Dermatology Foundation, Melanoma Research Foundation, and American Skin Association. S.L.B., P.D.A. and R.M. are supported by NIA P01 grant (P01AG031862). S.L.B. is also supported by NIH R01 CA078831. R.D.G. is supported by R01 GM106023 and the Progeria Research Foundation
Acute and rapid degradation of endogenous proteins by Trim-Away.
Protein depletion is a key approach to understanding the functions of a protein in a biological system. We recently developed the Trim-Away approach in order to rapidly degrade endogenous proteins without prior modification. Trim-Away is based on the ubiquitin ligase and Fc receptor TRIM21, which recognizes antibody-bound proteins and targets them for degradation by the proteasome. In a typical Trim-Away experiment, protein degradation is achieved in three steps: first, introduction of an antibody against the target protein; second, recruitment of endogenous or exogenous/overexpressed TRIM21 to the antibody-bound target protein; and third, proteasome-mediated degradation of the target protein, antibody and TRIM21 complex. Protein degradation by Trim-Away is acute and rapid, with half-lives of ~10-20 min. The major advantages of Trim-Away over other protein degradation methods are that it can be applied to any endogenous protein without prior modification; that it uses conventional antibodies that are widely available; and that it can be applied to a wide range of cell types, including nondividing primary human cells, for which other loss-of-function assays are challenging. In this protocol, we describe the detailed procedures for antibody preparation and delivery in mouse oocytes and cultured cells via microinjection and electroporation. In addition, we provide recommendations for antibody selection and validation, and for the generation of TRIM21-overexpressing cell lines for cases in which endogenous TRIM21 is limited. A typical Trim-Away experiment takes just a few hours.The research leading to these results received financial support from the Medical Research Council (MC_U105192711 and MC_U105181010), the Max Planck Society, the European Community’s Seventh Framework Programme (FP7/2007–2013) under grant agreement no. 241548, European Research Council (ERC) Starting Grant no. 337415 and a Wellcome Trust Investigator Award