As14 2769

Protein extractions were performed from de-achenized receptacles grounded in liquid nitrogen. In all cases, 600μl of extraction buffer [Tris-HCl 50 mM pH 7,5, Nonidet N-40, P9599 protease inhibitors (Sigma) and PMSF 1 mM] was mixed with 300 mg of tissue powder and incubated for 10 min on ice with eventual vortex. Samples were sonicated for 10 min on an ice-cold water bath and filtered through one-layer miracloth. Proteins were quantified by Bradford assay with Bio-Rad protein assay (Bio-Rad, 5000001) and 10 μg of protein were loaded into SDS-PAGE gels. For ATG8 and NBR1 detection, 15 or 8% acrylamide/bis-acrylamide resolving gels were used, respectively. Proteins were transfered onto polyvinylidene difluoride membranes (Merck-Millipore). AntiATG8 raised against Chlamydomonas reinhardtii (Agrisera, AS14 2769) and antiNBR1 raised against Arabidopsis thaliana NBR1 (Agrisera, AS14 2805) were used at 1:2,000 dilution. Secondary antibody was used at 1:5,000 dilution. Blots were developed using ECL substrate (Thermo Fisher Scientific). The developing reaction was detected using a Chemidoc XRS+ (Bio-Rad). Band quantification was performed with Image Lab software (Bio-rad).

Proteins were prepared from leaves collected from BPMV-0 and BPMV-GmATG2 plants treated in the dark for different time. The immunoblots were performed as previously described [68 (link)]. Anti-Ubq (Agrisera, AS08307) and anti-ATG8 (Agrisera, AS142769) were used for Western blotting.

A fresh leaf sample was macerated in liquid N₂ and homogenized in a buffer containing 50-mM Tris–HCl pH 8.0, 150-mM NaCl, 1-mM phenyl-methanesulfonyl fluoride, and 10-mM iodoacetamide) (Chung et al., 2009 (link)) and protease inhibitor cocktail. For immunoblot analysis, 15% of SDS-PAGE gel was prepared with 6-M urea (Wang et al., 2019 (link)). Following electrophoresis of the samples, the protein-containing SDS-PAGE gel was transferred to a nitrocellulose membrane. The levels of ATG8 and ATG8-PE were determined using an anti-ATG8 antibody (AS14 2769, Agrisera, Sweden) in 1:500 dilution. Histone H3 was used as a loading control. Thus, for determining the His-H3 level, anti-histone-H3 (AS10710, Agrisera, Sweden) was used as primary antibody in 1:2000 dilution. An anti-rabbit antibody conjugated to HRP was used as the secondary antibody.