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

•A gel mobility shift assay showed that oxidized PTEN migrated more rapidly than reduced PTEN on a non-reducing SDS–PAGE gel.•Supplementation of N-ethylmaleimide in the cell lysis buffer was critical for the apparent bands of oxidized and reduced PTEN.•These results show that the mobility shift assay is a convenient method to analyze the redox state of PTEN in cells. 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 Cys124 or nearby Cys71 was replaced with Ser suggesting that Cys124 and Cys71 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.