Measuring p66Shc Signaling Pathway Activation and Mitochondrial Translocation in Cultured Cells

Curr Protoc Toxicol. 2015 Nov 2;66:25.6.1-25.6.21. doi: 10.1002/0471140856.tx2506s66.

Abstract

The adaptor protein p66Shc links membrane receptors to intracellular signaling pathways, with downstream consequences on mitochondrial metabolism and reactive oxygen species production. Moreover, p66Shc has also been implicated in cancer development, progression, and metastasis. Increased phosphorylation of serine 36 residue of p66Shc very often correlates with oxidative stress-associated pathologies. The pro-oxidative role of p66Shc also appears to be involved in chemical toxicity, being an important component of stress responses triggered by xenobiotics. Here, we present a protocol that can be used: (a) for isolation of mitochondrial, cytosolic, and mitochondrial-associated membrane fractions from adherent cells lines; (b) to perform p66Shc detection with specific antibodies in order to monitor its translocation between different cellular compartments in response to the oxidative stress; and (c) to modulate the p66Shc pathway with the use of pharmacological approaches or gene-silencing methods.

Keywords: cell signaling pathways; mitochondria; oxidative stress; protein phosphorylation and p66Shc protein.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cells, Cultured
  • Endoplasmic Reticulum / metabolism
  • Humans
  • Mitochondria / drug effects
  • Mitochondria / metabolism*
  • Mitochondrial Membranes / drug effects
  • Mitochondrial Membranes / metabolism*
  • Oxidative Stress / drug effects
  • Protein Transport
  • Shc Signaling Adaptor Proteins / genetics
  • Shc Signaling Adaptor Proteins / metabolism*
  • Signal Transduction / drug effects*
  • Src Homology 2 Domain-Containing, Transforming Protein 1
  • Xenobiotics / toxicity

Substances

  • SHC1 protein, human
  • Shc Signaling Adaptor Proteins
  • Src Homology 2 Domain-Containing, Transforming Protein 1
  • Xenobiotics