Quabit system

The analysis was performed according to the procedure outlined by Bello et al. (2014) with minor modifications. Initially, the degradation buffer (25 mM Tris-HCl, pH 7.5,10 mM NaCl, 10 mM MgCl2, 4 mM PMSF, 5 mM DTT, and 10 mM ATP) was used to extract the total protein from 10-day-old rice seedlings. The extracted protein was quantified by using a Quabit system (Invitrogen, Carlsbad, CA). Then purified recombinant proteins, 5 μg for each protein sample, were incubated with the extracted total proteins (200 μg) in the degradation buffer (20 μL) at 28 °C. Samples were collected at specified time intervals, and the degradation reactions were stopped immediately. The amount of the target protein was determined by Western blotting using anti-His, anti-Flag and anti-MYC antibodies.

The experiment was conducted by following Lv et al., 2014. Briefly, total protein of 10 days‐after‐germination rice seedlings were extracted in degradation buffer (25 mm Tris‐HCl, pH 7.5, 10 mm NaCl, 10 mm MgCl₂, 4 mm PMSF, 5 mm DTT, and 10 mm ATP) and quantified using a Quabit system (Invitrogen, Carlsbad, CA). Purified recombinant proteins (5 μg of each) were incubated with 200 μg extracted total proteins in 20 μL degradation buffer at 28 °C. Reactions were terminated at indicated time points, and the protein abundance was visualized by immune detection against anti‐HIS. The immune signals were quantified using Quantity Tools of Image Lab software (Bio‐Rad, Hercules, CA). The half‐life of HIS‐NF‐YB1 was calculated based on the degradation curves deduced from the tested time points. The protein intensities were quantified using ImageJ software with triplicates. The dissociation‐one phase exponential decay curve was plotted on a semilog graph using Graphpad Prism (5.0) software as previously described by (Lv et al., 2014).