Total proteins were extracted from N. benthamiana leaves 2 days after infiltration. Immunoprecipitation experiments were performed using N. benthamiana leaves 2-days after infiltration. Tissues were ground in liquid nitrogen and resuspended in ice-cold RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors and 20mM N-ethylmaleimide to inhibit deubiquitinases during protein preparation. Samples were solubilized at 4°C for 30 minutes followed by centrifugation for 15 min at 14,000 x g at 4°C to eliminate cell debris. Supernatants were subjected to immunoprecipitation with the GFP-Trap Magnetic beads (ChromoTek). Immunoprecipitates were eluted off beads using Laemmli buffer. Samples were boiled for 5 minutes at 95°C and migrated on a 10% Bis-Tris NuPage Gel (Life Technologies).
Total proteins or immunoprecipitates were separated on a 10% Bis-Tris NuPage Gel (Life Technologies) and transferred onto a nitrocellulose membrane. Proteins were detected using the following antibodies: Monoclonal anti-GFP horseradish peroxidase-coupled (Miltenyi Biotech 130-091-833, 1/5,000), anti-ubiquitin P4D1 (Cell Signaling, 1/2,000). Experiments were done in triplicates. Rubisco was used as loading control.
Total proteins or immunoprecipitates were separated on a 10% Bis-Tris NuPage Gel (Life Technologies) and transferred onto a nitrocellulose membrane. Proteins were detected using the following antibodies: Monoclonal anti-GFP horseradish peroxidase-coupled (Miltenyi Biotech 130-091-833, 1/5,000), anti-ubiquitin P4D1 (Cell Signaling, 1/2,000). Experiments were done in triplicates. Rubisco was used as loading control.