Protein extractions of SOD, CAT, glutathione peroxidase (GPX; E.C.
1.11.1.9) and GR were performed using 4X PEB (protein extraction buffer,
AS08 300 Agrisera Inc.). Samples were dissolved in an equal volume of
sample buffer (2X Laemmli sample buffer 1610737 Bio-Rad) and heated at
95 °C for 5 min. Separation of total proteins (30 μg) by 12% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, (TGX
Stain-Free Precast Protein Gels, 4568095 Bio-Rad) was performed by
electrophoresis (Mini-PROTEAN Tetra Cell system, 165800 Bio- Rad) at
room temperature. The marker (Precision Plus Protein Western C Blotting
Standards, 1610376 Bio-Rad) was used to determine the molecular weight
of the proteins. Proteins separated in gel electrophoresis were
transferred to the PVDF membrane (Trans-Blot Turbo Mini PVDF Transfer
Packs, 1704156 Bio-Rad) using the Trans-Blot Turbo Transfer System
(1704155 Bio-Rad). After transfer, the membrane was blocked at
4 °C with 2.5% milk powder in
TBS. Fe-SOD Chloroplastic Fe-Dependent Superoxide Dismutase (A S06 125)
for SOD, Glutathione Peroxidase Chloroplastic (A S04 055) for GPX, and,
Catalase (A S09 501) for CAT were primary used antibodies and they
incubated overnight at 4 °C. After incubation, the membranes were
incubated with goat anti-rabbit IgG-HRP secondary antibodies (AS09 602
Agrisera) for 4 h at room temperature. The density of the scanned
protein bands was calculated with Image Lab Software (1709690, Bio-Rad).