Protease inhibitor cocktail for plant cells

The enzymatic activities were determined as described by Santangeli et al. [28 (link)]. Frozen homogenized samples, with polyvinylpolypyrrolidone (PVPP), were suspended in 1% PBS with protease inhibitor cocktail for plant cells (Sigma), incubated overnight at 4 °C, centrifuged at 10,000× g at 4 °C for 30 min and the supernatants were recovered and stored at −20 °C until analysis. Superoxide dismutase (SOD) (EC 1.15.1.1) activity was assayed by NPAGE (native polyacrylamide gel electrophoresis). Samples (40 μg of proteins) were loaded and separated on native polyacrylamide gels. SOD activity was visualized following the procedure described by Beauchamp and Fridovich [71 (link)]. SOD activity was expressed as Arbitrary Units (A.U.), which corresponds to the pixel density of each lane obtained by the program Image J 1.53A.

FSL0260 beads were prepared as previously described^{47 (link)}. We incubated 150 µg sonicated potato mitochondria with FSL0260 beads in 1 mL binding buffer containing 50 mM Tris HCl (pH 7.5), 150 mM NaCl, 0.5% Tween 20, 0.1 mM EDTA and Protease Inhibitor Cocktail for plant cells (Sigma-Aldrich St. Louis, USA) for 3 h at 4 °C. The beads were then washed three times in 1 mL binding buffer and eluted with an SDS sample buffer. The eluted protein was then subjected to SDS-PAGE.