Heat treatment triggered the proteolytic modification of Pho1 by the 20S proteasome.

<p>In two parallel experiments sweet potato root discs (0.5 mm thick) were pretreated without (Control) or with 100 µM of MG132 (+MG132), and then incubated at 45°C for the indicated time periods. BA, beta amylase; Pho1, intact Pho1; Pho1_d, proteolytic modified Pho1. (<b>A</b>) The crude protein extracts of the discs (10 µg) were separated by 7.5% native PAGE and analyzed by Coomassie staining, Pho1 activity staining or western blotting with H7c, αL78 or α20S antibody, respectively. Data are representative of three independent experiments. (<b>B</b>) Quantitative densitometry of the protein levels of intact Pho1 on the western blots detected by αL78 antibody in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035336#pone-0035336-g005" target="_blank">Figure 5A</a>. The intact Pho1 level of the sample pretreated without (Control) or with MG132 (+MG132) at time zero was expressed as 100%. Values are means ± S.D. from three independent experiments. (<b>C</b>) The 20S proteasome activities in the samples pretreated without (Control, bars in white) or with MG132 (+MG132, bars in black) were also determined by using Suc-LLVY-AMC as the substrate. The 20S proteasome activity in the control sample at time zero was expressed as 100%. Values are means ± S.D. from three independent experiments.