Assessment of ESCRT Protein CHMP5 Activity on Client Protein Ubiquitination by Immunoprecipitation and Western Blotting

Francheska Son; Katharine Umphred-Wilson; Jae-Hyuck Shim; Stanley Adoro

doi:10.1007/978-1-4939-9492-2_16

Assessment of ESCRT Protein CHMP5 Activity on Client Protein Ubiquitination by Immunoprecipitation and Western Blotting

Methods Mol Biol. 2019:1998:219-226. doi: 10.1007/978-1-4939-9492-2_16.

Authors

Francheska Son¹, Katharine Umphred-Wilson¹, Jae-Hyuck Shim², Stanley Adoro³

Affiliations

¹ Department of Pathology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA.
² Division of Rheumatology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA.
³ Department of Pathology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA. sxa726@case.edu.

Abstract

The charged multivesicular body protein-5 (CHMP5) is a member of the endosomal-sorting complex required for transport (ESCRT) that controls membrane-scission events in eukaryotic cells. Recent studies have revealed novel functions of CHMP5 beyond its role in the ESCRT machinery, notably as a critical nonenzymatic regulator of the ubiquitination and subsequent degradation of proteins in immune cells. Here we describe an immunoprecipitation and western blot methodology for assessing CHMP5 activity on client protein ubiquitination in T lymphocytes.

Keywords: CHMP5; Deubiquitinase; Posttranslational modification; T lymphocytes; Ubiquitination.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Blotting, Western / methods*
Endosomal Sorting Complexes Required for Transport / metabolism*
Endosomes / metabolism
HEK293 Cells
Humans
Immunoprecipitation / methods*
Jurkat Cells
T-Lymphocytes
Ubiquitination*

Substances

CHMP5 protein, human
Endosomal Sorting Complexes Required for Transport

Abstract

Publication types

MeSH terms

Substances

Grants and funding