Figure 3

More efficient degradation of oxidized proteins by the 20S immunoproteasome (IP) and the two 20S intermediate proteasomes β5i (SIP) and β1i­-β5i (DIP). (a–c) Western blot analysis of the kinetics of degradation of (a) native and (b) oxidized calmodulin by the four 20S proteasome subtypes. (c) Densitometric evaluation of the kinetics of the degradation of the oxidized calmodulin by the four proteasome subtypes. All values (+ SEM) are collected from seven independent experiments using three different batches of purified proteasomes. (d, e) Kinetics of degradation of tritium-labeled (d) native and (e) oxidized hemoglobin. These two forms of hemoglobin were incubated with the four 20S proteasome subtypes. To monitor protein degradation, samples were collected at different time points, precipitated with trichloroacetic acid (TCA) and the radioactivity present in the supernatant was measured. Remaining hemoglobin was quantified by subtracting the radioactivity measured in the supernatant from the total radioactivity. Proteins were oxidized by 24 h incubation in 50 mM H₂O₂. All values are means (+ SEM) of seven independent experiments using three different batches of purified proteasomes. Full-length images for (a-b) are presented in Fig. S11.