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).