Organelle Separation and Cell Signaling

Methods Mol Biol. 2017;1557:111-115. doi: 10.1007/978-1-4939-6780-3_11.


Recent findings indicate that some signaling hubs coalesce at the surfaces of organelles through the accumulation of ubiquitylated components required for the signal transduction. For instance, ubiquitylated components of the NF-κB pathway accumulated at the endoplasmic reticulum while ubiquitylated components of the IRF3 pathway are found at the Golgi apparatus. Here we describe simple methods to observe and assess these ubiquitylated components by immunoblotting using differential centrifugation and in vitro assays.

Keywords: Cell fractionation; Differential centrifugation; Immunoblotting; Signaling; Ubiquitination.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Western
  • Cell Fractionation* / methods
  • Centrifugation, Density Gradient / methods
  • Interferon Regulatory Factor-3 / metabolism
  • NF-kappa B / metabolism
  • Organelles / metabolism*
  • Phosphorylation
  • Signal Transduction*
  • Ubiquitination


  • Interferon Regulatory Factor-3
  • NF-kappa B