title: Assay of the redox state of the tumor suppressor PTEN by mobility shift.
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.
authors: Department of Biochemistry, Department of Biomedical Science, Research Center for Aging and Geriatrics, Research Institute of Medical Sciences, Chonnam National University Medical School, Gwangju 501-190, Republic of Korea; School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea.
authors: Seong-Jeong
authors: SJ
authors: Han
authors: Department of Pediatrics and Clinical Neuroscience, Alberta Children's Hospital Research Institute for Child and Maternal Health, Faculty of Medicine, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta T2N4N1, Canada.
authors: Younghee
authors: Y
authors: Ahn
authors: Department of Biochemistry, Department of Biomedical Science, Research Center for Aging and Geriatrics, Research Institute of Medical Sciences, Chonnam National University Medical School, Gwangju 501-190, Republic of Korea.
authors: Iha
authors: I
authors: Park
authors: Department of Biochemistry, Department of Biomedical Science, Research Center for Aging and Geriatrics, Research Institute of Medical Sciences, Chonnam National University Medical School, Gwangju 501-190, Republic of Korea.
authors: Ying
authors: Y
authors: Zhang
authors: Department of Biochemistry, Department of Biomedical Science, Research Center for Aging and Geriatrics, Research Institute of Medical Sciences, Chonnam National University Medical School, Gwangju 501-190, Republic of Korea.
authors: Inyoung
authors: I
authors: Kim
authors: Department of Biochemistry, Department of Biomedical Science, Research Center for Aging and Geriatrics, Research Institute of Medical Sciences, Chonnam National University Medical School, Gwangju 501-190, Republic of Korea.
authors: Hyun Woo
authors: HW
authors: Kim
authors: COTDE Inc. 19-3, Ugakgol-gil, Susin-myeon, Cheonan-si, Chungcheongnam-do 330-882, Republic of Korea.
authors: Chang-Sub
authors: CS
authors: Ku
authors: Department of Biochemistry, Department of Biomedical Science, Research Center for Aging and Geriatrics, Research Institute of Medical Sciences, Chonnam National University Medical School, Gwangju 501-190, Republic of Korea.
authors: Kee-Oh
authors: KO
authors: Chay
authors: Department of Biochemistry, Department of Biomedical Science, Research Center for Aging and Geriatrics, Research Institute of Medical Sciences, Chonnam National University Medical School, Gwangju 501-190, Republic of Korea.
authors: Sung Yeul
authors: SY
authors: Yang
authors: Department of Biochemistry, Department of Biomedical Science, Research Center for Aging and Geriatrics, Research Institute of Medical Sciences, Chonnam National University Medical School, Gwangju 501-190, Republic of Korea.
authors: Bong Whan
authors: BW
authors: Ahn
authors: COTDE Inc. 19-3, Ugakgol-gil, Susin-myeon, Cheonan-si, Chungcheongnam-do 330-882, Republic of Korea.
authors: Dong Il
authors: DI
authors: Jang
authors: Department of Biochemistry, Department of Biomedical Science, Research Center for Aging and Geriatrics, Research Institute of Medical Sciences, Chonnam National University Medical School, Gwangju 501-190, Republic of Korea. Electronic address: leesr@chonnam.ac.kr.
authors: Seung-Rock
authors: SR
authors: Lee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D010084:Oxidation-Reduction; D051059:PTEN Phosphohydrolase / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D025521:Tumor Suppressor Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*
chemical_list: D025521:Tumor Suppressor Proteins; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human
keywords: Mobility shift assay; N-Ethylmaleimide; PTEN; Redox regulation