In vitro Degradation Assay

The in vitro degradation assay was carried out as previously described (Park et al., 2008). In brief, plant extracts were prepared from 10-day-old wild-type seedlings and resuspended in a cell-free degradation assay buffer (25 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 5 mM DTT, 10 mM NaCl, and 10 mM ATP). For degradation of the His-AtYY1, His-S284D and His-S284A recombinant proteins, cell debris was removed by centrifugation before adding to the proteins. The reaction mixtures (total reaction volume 110 μL) containing 300 μg plant extracts and 5 μg recombinant protein were incubated at 25°C, and at different time points (0, 15, 30, 50, or 100 min), 20 μL of the reaction mixture was transferred into new tubes containing 4 μL of 6× Protein Loading Buffer to stop the degradation process. Then, the samples were boiled for 3 min, separated on a 12% SDS-PAGE gel and detected with immunoblot analysis using anti-His antibodies (Abmart, Shanghai, China).