Extraction of Total Plant Protein and Western Blotting

Arabidopsis plants were immediately frozen upon harvesting and ground to a fine powder in liquid nitrogen using a pestle and mortar. A total of 0.5 mL of protein extraction buffer (50 mM Tris–HCl pH 7.5, 10% glycerol, 150 mM NaCl, 10 mM MgCl₂, 5 mM EDTA, 5 mM DTT, and 1 × protease inhibitors) was added to each sample and incubated at 4°C for 1 h with constant rotating. The homogenate was centrifuged at least two times at 13,000 × g for 10 min and the supernatant was collected after each centrifugation. The protein content was measured using the Pierce BCA protein assay kit (Thermo-Scientific). Western blot analysis of total protein run on a 10% native-PAGE was performed to investigate the conformational changes in the AtAPX1 protein. Polyclonal mouse anti-AtAPX1 antibody was used to detect AtAPX1 protein complexes from total protein extracts. Native-PAGE was performed as previously described (Moon et al., 2005).