Fig. 6.
Diurnal pattern of overoxidation of chloroplast 2-Cys Prxs from Arabidopsis. Arabidopsis wild-type seedlings that were grown under standard long-day conditions for 10 days were incubated during two consecutive days, the second one with continuous light, and samples were harvested at 4-h intervals, as indicated. (A) Protein extracts (12.5 µg of protein) were subjected to SDS-PAGE (12% acrylamide) supplemented with 4M urea under non-reducing conditions, electrotransferred onto nitrocellulose filters, and probed with the anti-2-Cys Prx antibody. (B) To determine the amount of overoxidized 2-Cys Prxs 60 µg of protein extracts were subjected to SDS-PAGE (12% acrylamide), supplemented with 4M urea under reducing conditions, electrotransferred onto nitrocellulose filters and probed with the anti-SO2/3 antibody. For each time point, band intensity was quantified with the Scion Image analysis software (Scion Corporation) and normalized against that of the indicated band (arrow) in the ponceau S-stained membrane, which is shown as a loading control. Relative densities refer to the intensity of the wild type, which was set at 1. dim, dimer; mon, monomer.