Fig. 2

Oxidative and salt stresses change the stoichiometry of proteasome complexes and alter activities of CP. A, B Anti-PBA1 immunoblot and Suc-LLVY-AMC (100 μM) stain of total lysates resolved on 4% Native-PAGE showing abundance and peptidase activity of the proteasome particles, respectively. PAG1-FLAG seedlings were stress-treated for 12 and 24 h before lysis in extraction buffers with (A) and without (B) 20 mM ATP. Loading was normalized based on fresh weight and a Ponceau S stain is included as a loading control. 0.02% SDS was used to artificially activate the free 20S CP. Three biological replicates were conducted, and one is shown. Bar graphs show digital quantifications of band intensities from immunoblots or activity gels. Data is expressed relative to the control for each time point, which is arbitrarily assigned a total signal value of 1.0. For Native-PAGE run with exogenous ATP, quantifications of RP₂CP, RP₁CP, and free CP are expressed as a fraction of total sample signal within stacked bars. All bar charts display the means of three biological replicates ± SEM. Significant differences in RP₂CP, RP₁CP or free CP signal between control and stress conditions were determined by independent Student’s t-tests (* = p < 0.05, ** = p < 0.01). It should be noted that physiological level of ATP is required to maintain the association of RP to CP. In B only trace RP1CP and RP2CP signals were shown in this representative Western blotting image and this particular exposure was chosen to best maintain the free CP signals in linear range