Redox control of Trx–ASK1 interaction

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Mammalian thioredoxin is a direct inhibitor of apoptosis signal‐regulating kinase (ASK) 1 Redox control of Trx–ASK1 interaction. (A) Binding of in vitro‐translated 35S‐ASK1 to GST fusion proteins. Purified GST fusion proteins immobilized on the beads were treated or untreated with 1 mM DTT on ice for 30 min, washed with a buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 10 mM EDTA, 1% Triton X‐100 and 1% deoxycholate and subjected to in vitro binding assay as described in Materials and methods. (B) Reducing condition‐dependent binding of Trx to ASK1. Purified GST–Trx immobilized on the beads was treated with the indicated concentration of DTT or β‐mercaptoethanol (β‐ME), and the binding was determined as described in (A) (top panel). To verify the amount of GST fusion proteins in the same sample, the lower part of the SDS–PAGE was cut out and immunoblotted with anti‐GST antibody (lower panel). (C) Oxidation‐induced dissociation of Trx from ASK1. Binding of in vitro‐translated 35S‐ASK1 to DTT‐treated GST–Trx and washing of beads were performed as described in (A). Thereafter, the beads were washed once with PBS, incubated in PBS with the indicated concentrations of H2O2 or diamide at 25°C for 15 min, washed twice with PBS, and then analyzed by SDS–PAGE with an image analyzer (top panel). To verify the amount of GST fusion proteins in the same sample, the lower part of the SDS–PAGE was cut out and immunoblotted with anti‐Trx antibody (lower panel). (D) Specific co–immunoprecipitation of endogenous Trx and transfected HA‐ASK1 by anti‐Trx antibody in 293 cells. Cell lysates were immunoprecipitated with normal goat serum (NGS), anti‐pyk2 goat antibody or anti‐Trx goat antibody and analyzed by immunoblotting as described in Materials and methods. (E) Effects of H2O2 and TNF‐α on the interaction of ASK1 with endogenous Trx. 293 cells transfected with HA‐ASK1 were treated with the indicated concentration of H2O2 or TNF‐α for 20 min and subjected to immunoprecipitation (by anti‐Trx antibody)‐immunoblotting analysis as in (D). (F) Inhibition of TNF‐α‐induced dissociation of Trx from ASK1 by Nac. 293 cells transfected with HA‐ASK1 were pretreated or not with 1 mM Nac for 1 h. The cells were then treated with the indicated concentration of TNF‐α for 20 min and subjected to immunoprecipitation (by anti‐Trx antibody)‐immunoblotting analysis as in (D). Top, copurified ASK1; middle, immunoprecipitated Trx; bottom, immunoblotting of aliquots of whole cell lysate by anti‐HA antibody. IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. EMBO J, Volume: 17, Issue: 9, Pages: 2596-2606, First published: 01 May 1998, DOI: (10.1093/emboj/17.9.2596)