Figure 4. Biochemical properties of the polymer form of oxidized DJ-1 detected in the erythrocytes of unmedicated PD patients.

(a) Proteins containing the polymer form of oxDJ-1 were separated by DEAE column chromatography, and the oxDJ-1 content of each fraction was determined by using competitive ELISA. The mean ± SD (n = 3) was shown. **P < 0.01, Tukey-Kramer test, ANOVA, when compared with buffer control. A major oxDJ-1 peak (Fr. 10) is indicated by the black arrow. (b) Proteins eluted in Fr. 10 by DEAE column chromatography were visualized by silver staining, and each band was subjected to in-gel digestion and MALDI-TOF MS analysis. Identified proteins are indicated. (c) Proteins eluted in Fr. 10 by DEAE column chromatography were subjected to western blot analysis for DJ-1, oxDJ-1, HSP90α, and proteasome subunit β type 4. (d) 20 S proteasome purified from human erythrocytes was incubated for 18 h at 4 °C in either the presence or absence of purified recombinant human oxDJ-1 in 50 mM HEPES buffer (pH 7.4) supplemented with 10% glycerol, 2 mM ATP and 2 mM DTT. Samples were then separated by gel chromatography, and each fraction was subjected to western blot analysis.