Western Blotting

For Western blotting, 200 mg of plant tissue of 35-day-old rice seedlings was ground in liquid nitrogen. The homogenate was resuspended for 60 min at 65°C in 1 ml extraction buffer [2% SDS, 12% sucrose, 50 mM dithiothreitol, 50 mM Na₂CO₃, 2 mM EDTA, pH 8.0, 0.1 mM phenylmethylsulfonylfluoride, and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA)]. The protein concentration was determined with the BCA Protein Assay kit (Thermo Scientific, Waltham, MA, USA). Antibodies against OsGUN4 (Li et al., 2014) was generated by BPI (Beijing Protein Innovation, Beijing, China) using the peptides FTRFFIRVGWMKKL. After separation by SDS-PAGE, the proteins were immunoblotted according to Li et al. (2014), using OsHSP (Os09g0482600) as a reference protein (Li et al., 2011).