Ubiquitination assay
For detection of ubiquitinated ApNMV 1a in vivo, Agrobacterium harboring
pCXSN-1a-HA construct or ApNMV infectious clone was transformed into
apple leaves of WT, MdBT2-OE , and MdBT2 MdBT2-anti,
respectively, via vaccum technique. The leaves were kept on MS medium
for five days and then collected for protein extraction after treated
with 50 μM MG132 for 10 h. The total protein was immunoprecipitated
using anti-HA antibody (Abmart) or anti-1a specific antibody using
BeaverBeads protein A/G immunoprecipitation Kit (BEAVER Biomedical
Engineering) per manufacture’s protocol. The precipitates were then
applied to western blot, and anti-HA, anti-1a, and anti-Ubi
(Sigma-Aldrich) antibodies were utilized to detect the target proteins
as described previously.
For detection the ubiquitinated ApNMV 1a in vitro, total protein were
first extracted from 35S::MdBT2-GFP transgenic apple calli pretreated
with 50 μM MG132 for 10 h. Then MdBT2-GFP active proteins were
precipitated by anti-GFP antibody (Abmart) using BeaverBeads protein A/G
immunoprecipitation Kit (BEAVER Biomedical Engineering) per
manufacture’s protocol. The ApNMV 1a-HIS protein obtained from
prokaryotic expression system was incubated with or without MdBT2-GFP
active proteins in incubation buffer at 30 °C for 12 h. The incubation
buffer consists of 50 mM Tris (pH 7.5), 2 mM dithiothreitol (DTT), 50 mM
MgCl2, 2 mM ATP, 100 ng human E1 (recombinant human His6
UBE1, BostonBiochem), 100 ng human E2 (recombinant human UbcH5b/UBE2D2,
BostonBiochem), and 1 μg ubi (recombinant human Myc Ubiquitin,
BostonBiochem). The target protein ubiquitination were detected using
the anti-HIS (Abmart) and anti-Ubi (Sigma-Aldrich) antibodies.