Preparation of the protein soluble fraction
Four-week-old hydroponically grown plants were either kept under control
conditions or treated with MgSO4 (10 and 40 mM) for 10,
30, and 180 min. Roots from 6 plants were used to prepare one sample.
The soluble fraction was prepared according to Janda et al.(2019). Briefly, roots were homogenized using homogenization buffer (50
mM HEPES-NaOH, pH 7.5, 0.4 M sucrose, 0.1 M KCl, 0.1 M
MgCl2) with a protease inhibitor cocktail (P 9599,
Sigma) and Pierce Phosphatase Inhibitor Mini Tablets (A23957, Thermo
Fisher Scientific). The homogenate was filtered and centrifuged at 6,000
× g for 15 min at 4°C, and the supernatant was centrifuged at 200,000 ×
g for 1 h at 4°C. The resulting supernatant (the soluble fraction) was
collected.