Total protein from approximately eight 8-d-old seedlings of Arabidopsis thaliana was extracted with NuPAGE sample buffer (106 mM Tris-HCl pH 8.5, 141 mM Tris Base, 2% lithium dodecyl sulfate, 0.51 mM EDTA pH 8.0, 10% glycerol, 0.22 mM Coomassie Blue G250, 0.166 mM Phenol Red) with 50 mM dithiothreitol and denatured at 100°C for 5 min. Proteins were separated by electrophoresis on a Bolt 10% Bis-Tris Plus gel (Invitrogen) and transferred for 40 min at 24 V to an Amersham Protran 0.45 µm nitrocellulose membrane (GE Healthcare Life Sciences) using semidry transfer. The membrane was blocked with 8% milk in TBS-T for 2 h at 4°C with agitation. The membrane was incubated in the primary antibody at a dilution of 1:4000 overnight at 4°C with agitation in 8% milk in TBS-T. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 3 times for 5 min in 8% milk in TBS-T at 4°C with agitation. The membrane was incubated in secondary antibody (horse radish peroxidase conjugated goat anti-rabbit IgG) diluted to 1:5000 in 8% milk in TBS-T for 4 h at 4°C with agitation. The membrane was washed 3 times for 5 min in TBS-T at 4°C with agitation and incubated for 2 min with WesternBright chemiluminescent detection reagent (Advansta). Exposure time was 30 seconds.

Courtesy of Pierce Young, Rice University, USA

NBR1 (Autophagy substrate NBR1) is involved in selective autophagy process of damaged organelles, intracellular microbes, protein aggregates, cellular structures and specific soluble proteins. NBR1 mediates the process as an autophagic adapter. The protein has two UBA domains but only the C-terminal UBA domain bound ubiquitin. Alternative names: At4g24690, Putative uncharacterized protein F22K18.110.