Bulletin of the Korean Chemical Society

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Dual-specificity Phosphatase 8 Promotes the Degradation of the Polyglutamine Protein Ataxin-1

  • Lee, Do Hee (College of Pharmacy, Chung-Ang University) ;
  • Cho, Sayeon (Department of Biotechnology, College of Natural Science, Seoul Women's University)
  • Received : 2013.10.01
  • Accepted : 2013.10.21
  • Published : 2014.01.20
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Abstract

Keywords

Experimental

Cell Culture and Transfection. Human embryonic kidney (HEK) 293T cells were maintained at 37 ℃ in DMEM supplemented with 10% fetal bovine serum and penicillin/streptomycin in the presence of 5% CO2. For transient transfection, 1.4 × 106 cells were plated in 60-mm cell culture plates, grown overnight, and transfected with various plasmid DNA using LipofectamineTM reagent (Invitrogen)

Immunoblot Analysis. After 48 h of expression of Xpress-ataxin-1 and FLAG-DUSP proteins (for experiments studying the effects of the catalytic mutant of DUSP8, HA-ataxin-1 and GST-DUSP8 were employed), HEK293T cells were collected and then lysed in NP-40 lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40) supplemented with Complete-MINITM protease inhibitor mixture (Roche) for 20 min at 4 ℃, followed by centrifugation at 13,000 × g for 20 min. Immunoblot analysis using anti-Xpress antibody (1:2,000; Invitrogen), anti-FLAG antibody (1:1,000; Sigma) or anti-GST antibody (1:1,000; Sigma) were performed to determine the relative amounts of individual proteins. The protein bands were visualized using ECL detection system (PIERCE).

Fractionation of Cell Lysate. To analyze the relative amounts of ataxin-1 and DUSP8 in the soluble and insoluble fractions, HEK293T cells expressing Xpress-tagged ataxin-1 with or without FLAG-tagged DUSP8 were lysed in NP-40 lysis buffer, clarified by centrifugation at 1,000 × g for 10 min and then subjected to the second round of centrifugation at 20,000 × g for 30 min. The supernatants were collected and referred as the soluble fraction. The pellets, referred as the insoluble fraction, were washed three times with NP-40 lysis buffer and solubilized with an equal volume of 1 × SDS sample buffer. The relative amounts of individual proteins were determined by western-blot analysis.

Filter Retardation Assay. Filter retardation assay was performed using the protocol described in a previous study.13 Cell lysate containing Xpress-tagged ataxin-1 was applied onto nitrocellulose membranes (0.2 μm; S & S) using a dot-blot microfiltration kit (Bio-Rad). The membranes were blocked in 5% non-fat dried milk in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) for 1 h and subsequently incubated with anti-Xpress antibody. After washing membranes with TBST, the presence of ataxin-1 in large aggregates was detected by using ECL detection system.

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