Protein blot analysis
Rice grains (from 5 to 25 DAF) were homogenized in TEDM buffer (20 mM
Tris/HCl, pH 7.5, 1 mM DTT, 5 mM EDTA, and 10 mM MgCl2)
containing a complete protease inhibitor cocktail (Roche). The
homogenate was centrifuged at 6,000 g for 10 min at 4°C to remove
cellular debris, and the supernatant was clarified by centrifugation at
12,000 g for 10 min at 4°C. The soluble proteins were separated by
SurePAGE (4-12%, Genscript) and blotted onto PVDF membrane (Millipore).
Anti-HSP82 antibodies were used as a loading control (Beijing Protein
Innovation).