Cell-free degradation assay
The 1a-HIS protein was obtained from prokaryotic expression system usingE. coli BL21 (DE3). Total proteins were extracted from apple calli using degradation buffer: 25 mM Tris-HCl (pH 7.5), 10 mM NaCl, 10 mM MgCl2, 5 mM DTT, 10 mM ATP, and 4 mM phenylmethysulfonyl fluoride (Wang et al., 2009). The concentration of collected protein supernatants and 1a-HIS were measured using the Bradford reagent (Bio-Rad). Then 100 ng 1a-HIS protein and 300 ng total proteins were mixed and incubated at 22 °C for the indicated time. For the proteasome inhibition assay, the mixed proteins were pretreated with proteasome inhibitor MG132 (50 μM) for 30 min, while dimethyl sulfoxide (DMSO) served as mock. Then protein mixture was collected at indicated time points and the reactions were stopped by SDS-PAGE loading buffer and boiled for 5 min. The samples were then applied to western blot assay for protein degradation analysis. The results were quantified using Quantity One 1-D Analysis software (Bio-Rad).