Endosperm Protein and Starch Extraction

The total protein extraction method was the same as described above. Protein and starch granules were extracted from the supernatant. Briefly, 60 mg of developmental endosperm was ground and mixed with a 200 μl of buffer (10 mM Tris–HCl, pH 8.0) solution and centrifuged at 8,000g for 5 min. Next, the supernatant was collected and centrifuged three times. The resulting pellet, containing starch granules, was then washed 10 times with the buffer solution. Protein isolation was performed using SDS-PAGE as described above, following which proteins were transferred to a polyvinylidene fluoride membrane. GBSSI proteins were detected using GBSSI antibodies from rabbit serum (ABclonal, Anhui, China) at a dilution of 1:1,000 as the primary antibody. Horseradish peroxidase-labeled goat anti-rabbit IgG(H + L) (11000; Beyotime, Shanghai, China) was used as the secondary antibody. The blots were visualized using BeyoECL Star (Beyotime, Shanghai, China; Hebelstrup et al., 2017 (link)).