Fig. 3

Cellular ATP contents and extrinsic proteasome-associated proteins are affected by oxidative and salt stresses. A, B Cellular levels of ATP, ADP (A) and ATP/ADP ratio (B) of PAG1-FLAG seedlings stress-treated for 24 h. Data are displayed as means ± SEM of six biological repeats. Significant differences between control and stress conditions were determined by independent Student’s t-tests (* = p < 0.05, ** = p < 0.01). C, D Co-immunoprecipitation (Co-IP) of PAG1-FLAG-tagged proteasomes from 12- and 24-h stress-treated seedlings via M2 anti-FLAG antibody resin. Cell lysis and co-IP were performed in the presence (C) and absence (D) of 20 mM ATP. Eluates are visualized on a silver-stained 12% SDS-PAGE gel. E Immunoblotting analysis with anti-HSP70, anti-FLAG, anti-RPN1, and anti-PA200 antibodies performed on Co-IP eluates from the 12-h time point shown in (C, D). For the immunoblots, Co-IP eluates were loaded alongside increasing concentrations of total lysate (TL) obtained from the 12-h control (C) sample (+ATP)