Assay of the redox state of the tumor suppressor PTEN by mobility shift

Methods. 2015 May:77-78:58-62. doi: 10.1016/j.ymeth.2015.01.007. Epub 2015 Jan 27.

Abstract

PTEN is reversibly oxidized in various cells by exogenous hydrogen peroxide as well as by endogenous hydrogen peroxide generated when cells are stimulated with growth factors, cytokines and hormones. A gel mobility shift assay showed that oxidized PTEN migrated more rapidly than reduced PTEN on a non-reducing SDS-PAGE gel. Oxidized PTEN was reduced when treated with dithiothreitol. Supplementation of N-ethylmaleimide in the cell lysis buffer was critical for the apparent bands of oxidized and reduced PTEN. Formation of oxidized PTEN was abolished when the active site Cys(124) or nearby Cys(71) was replaced with Ser suggesting that Cys(124) and Cys(71) are involved in the formation of an intramolecular disulfide bond. These results show that the mobility shift assay is a convenient method to analyze the redox state of PTEN in cells.

Keywords: Mobility shift assay; N-Ethylmaleimide; PTEN; Redox regulation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Electrophoresis, Polyacrylamide Gel / methods*
  • HeLa Cells
  • Humans
  • Mice
  • Molecular Sequence Data
  • NIH 3T3 Cells
  • Oxidation-Reduction
  • PTEN Phosphohydrolase / analysis*
  • PTEN Phosphohydrolase / genetics
  • PTEN Phosphohydrolase / metabolism*
  • Rabbits
  • Tumor Suppressor Proteins / analysis*
  • Tumor Suppressor Proteins / genetics
  • Tumor Suppressor Proteins / metabolism*

Substances

  • Tumor Suppressor Proteins
  • PTEN Phosphohydrolase
  • PTEN protein, human