FIGURE 7.

Lack of effect of exposure to H₂O₂ and GSH on the composition of 20 S CP or 26 S proteasomes. A, purified 20 S CP or 26 S proteasomes were incubated with or without H₂O₂ and GSH and then resolved in Clear Native PAGE gels. Panels show the Coomassie-stained gel and Western blots stained for Rpn1 or Rpn2. B, 26 S proteasomal activity was determined after resolution of proteasomes in Clear Native PAGE followed by measuring proteasomal activity in the gel using Suc-Leu-Leu-Val-Tyr-AMC as a substrate. The left panel shows that equal amounts of the 26 S proteasome complexes were present in each sample as determined by staining with Coomassie Blue, whereas the right panel presents a representative AMC fluorescence image. RP, regulatory particle; CP, core particle. RP-CP single or RP(2x)-CP double regulatory particles are in complex with a single core particle. Representative gels are shown. A second experiment provided similar results.