Dilution series of recombinant 14-3-3 υ protein were spotted on nitrocellulose membrane, together with a dilution series of PRX-IIE as a calibration curve. The membrane was blocked with Tris-buffered saline (TBS), pH 7.5, containing 1% (w/v) milk powder. The membrane was incubated with PRX-IIE or its phospho-mimicry variants in TBS with either 1 mM DTT or 100 µM H2O2 overnight at 4 °C. After three times washing with TBS for 5 min, proteins were detected using a specific anti-PRX-IIE antibody, a peroxidase-labeled secondary antibody against rabbit, ECL® substrate (GE Healthcare, Chicago, IL, USA), and X-ray films. Intensity quantification of the spots after documentation was done using ImageJ. The relative amounts of bound PRX-IIE were determined in the linear range of the blots.

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