Hyperoxidation of ERp57 is intensified by removal of regulatory disulfide bonds in Ero1β (A–C) Where indicated, expression of Ero1β variants was induced.

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Hyperoxidation of ERp57 is intensified by removal of regulatory disulfide bonds in Ero1β (A–C) Where indicated, expression of Ero1β variants was induced with Dox for 24 h. Hyperoxidation of ERp57 is intensified by removal of regulatory disulfide bonds in Ero1β (A–C) Where indicated, expression of Ero1β variants was induced with Dox for 24 h. Prior to lysis, cells were treated with NEM to alkylate free thiols. After cell lysis, cysteines present in disulfides were reduced and decorated with AMS. Such AMS modification of active-site cysteines originally present in the oxidized state gives rise to slower SDS–PAGE mobility compared with the (NEM-decorated) pool of ERp57 containing reduced active-site cysteines. The cellular redox state of ERp57 was visualized by Western blotting. DTT and Diamide (Dia) treated-cells were used to show the mobility of fully oxidized (Ox) and reduced (Red) ERp57. A vertical hairline denotes removal of lanes. Henning G. Hansen et al. Biosci. Rep. 2014;34:e00103 ©2014 by Portland Press Ltd