Immunoblotting of Endogenous COP1 Protein

To detect endogenous COP1 protein levels, an anti-COP1 polyclonal antibody was used (Lee et al., 2017 (link)). Fresh seeds (harvested within 1 month before use) were imbibed in distilled water with or without 10 μM GA₃ or 10 μM PAC, and harvested at each time point. Total crude extracts were prepared using extraction buffer containing 50 mM Tris–HCl pH 7.5, 4 M urea, 150 mM NaCl, 1 mM EDTA, and protease and phosphatase inhibitor mixtures (1 mM PMSF, 5 μg/mL leupeptin, 5 μg/mL aprotinin, 5 μg/mL pepstatin, 5 μg/mL antipain, 5 μg/mL chymostatin, 2 mM Na₂VO₃, 2 mM NaF, and 50 μM MG132), separated by SDS-PAGE, and then immunoblotted with anti-COP1, and anti-Histone 3 (Abcam) antibodies.