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.


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*


  • CHMP5 protein, human
  • Endosomal Sorting Complexes Required for Transport