Samples were heat-denatured and separated via SDS-PAGE, followed by transfer to polyvinylidene fluoride membranes (Immobilon-P) (Merck Millipore, Watford, UK). Membranes were blocked with Blocking One (Nacalai Tesque) or Tris-buffered saline-Tween (TTBS) containing 3% skim milk (Nacalai Tesque), after which they were incubated with antibodies in TTBS containing 5% skim milk overnight at 4°C. Antibodies used in this study included the following: anti-DDDDK (FLAG)-tag (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan), anti–β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), anti-ADH5 prepared as we described elsewhere (29 (link)), anti-protein S-glutathionylation (Virogen, Watertown, MA), anti-yeast 3-phosphoglycerate kinase (Pgk1) (clone: 22C5D8; Abcam, Cambridge, UK), and anti-HA (Medical & Biological Laboratories Co., Ltd.). Membranes were washed three times with TTBS and then incubated with a horseradish peroxidase–conjugated secondary antibody for 1 hour at room temperature. After the membranes were washed again three times with TTBS, immunoreactive bands were detected via a chemiluminescence reagent (ECL Prime Western Blotting Detection Reagent; GE Healthcare) with a luminescent image analyzer (ImageQuant LAS 500; GE Healthcare).