F1804

For Western blots, the
following primary antibodies were diluted in BSA (5%) in 1× PBS
buffer: monoclonal ANTI-FLAG M2 antibody produced in mouse (1:1000,
F1804, Merck); anti-β-actin antibody, mouse monoclonal (1:2000,
A1978, Merck). HRP goat antirabbit IgG antibody (peroxidase) (1:3000,
PI-1000, Vectorlabs), and peroxidase antimouse IgG (H+L) (affinity
purified) (1:3000, PI-2000, Vectorlabs) were used as secondary antibodies.
For IF assays, the following primary antibodies were diluted in
blocking buffer (FBS 3% in 1× PBS): the 5-hydroxymethylcytosine
(^5hmC) antibody (pAb) (1:500, Active Motif cat#: 39769)
and monoclonal ANTI-FLAG M2 antibody produced in mouse (1:500, F1804,
Merck). Goat antimouse Alexa Fluor 568 (1:500, A-11031, Invitrogen)
and Goat antirabbit Alexa Fluor 647 (1:500, A-21236, Invitrogen) were
used as secondary antibodies.

HEK293T cells were co-transfected with Flag-NEP truncation mutants and HA-CRM1. Transient transfected cells were washed twice with PBS and lysed in NP-40 lysis buffer (50 mM Tris-HC1, pH 7.4.150 mM NaC1.1% NP-40, sodium pyrophosphate, β-glycerophosphate, sodium orthovanadate, sodium fluoride, EDTA, leupeptin) supplemented with protease inhibitor (Beyotime). Whole-cell lysate was firstly precleared with protein A/G slurry (D2117; Santa Cruz). After centrifugation at 1000× g for 5 min at 4 °C, supernatant was incubated with 1 μg of mouse anti-Flag MAb (F1804; Sigma-Aldrich) and 20 μL of protein A/G slurry (D2117; Santa cruz) and incubated with rotation for 4 h at 4 °C. Immunoprecipitated samples collected by centrifugation were washed with NP-40 lysis buffer four times. The final pellet was dissolved in 2× SDS loading buffer for SDS-PAGE and Western blotting. Immunoprecipitations and the whole-cell lysates were probed with mouse anti-Flag MAb (F1804; Sigma-Aldrich), rabbit anti-Flag PcAb (9121; HUABIO), and mouse anti-HA MAb (H9658; Sigma-Aldrich).

The in vitro ubiquitylation assay was carried out as described previously^{36 (link)}. In brief, the MBP–ZnF-CT and His–-BKI1 recombinant proteins were expressed and purified from Escherichia coli strain BL21 (DE3). MBP–ZnF-CT alone, or together with His–BKI1, was incubated with E1 (UBA1–GST, 50 ng), E2 (UBC10–GST, 50 ng) and Flag–ubiquitin (1 µg) in a reaction buffer (50 mM Tris-HCl pH 7.5, 5 mM ATP, 20 mM MgCl₂ and 1 mM dithiothreitol) at 30 °C for 1.5 h. Similarly, for the assay of ZnF-mediated TaBKI1 ubiquitylation, TaBKI1 at different concentrations was incubated with MBP–ZnF-CT, E1 (UBA1–GST, 50 ng), E2 (UBC10–GST, 50 ng) and Flag–ubiquitin (1 µg) in reaction buffer. The reaction was stopped by adding SDS loading buffer. The obtained samples were boiled at 100 °C for 7 min, and the proteins were detected with anti-Flag (1:2,000 dilution, F1804, Sigma) antibody using SDS–PAGE. For the in vivo ubiquitylation assay, TaBKI1–GFP, ZnF–MYC and Flag–ubiquitin were co-expressed in N. benthamiana leaf epidermal cells. TaBKI1–GFP proteins were immunoprecipitated and purified to ubiquitin-conjugated TaBKI1–GFP through immunoblotting with anti-Flag (1:2,000 dilution, F1804, Sigma) antibody using anti-GFP magnetic agarose beads (gtma-20, Chromotek).