Chloroplast and nuclear isolation, protein extraction, and western blotting

To extract total protein from tobacco leaves, frozen samples were ground into powder and suspended in protein extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, 0.2% Nonidet P-40 (NP-40) containing 50 μg/ml of tosyl-L-phenylalaninyl-chloromethylketone, 50 μg/ml of tosyl-L-lysine-chloromethylketone, serine protease inhibitors, 0.6 mM phenylmethylsulfonyl fluoride (PMSF), 80 μM MG115, 80 μM MG132, and complete protease inhibitor cocktail tablet (Roche, USA)²⁰.

To isolate chloroplast stroma proteins from tobacco leaves, chloroplasts were first isolated from leaves using a chloroplast isolation kit (Sigma-Aldrich, USA), after which intact chloroplasts were harvested by 40/80% Percoll gradient. Intact chloroplasts were suspended in chloroplast lysis buffer (0.5 mM HEPES-KOH, pH 7.5, 2 mM MgCl₂, 1 mM NaF, 1 mM EDTA, 1 mM PMSF, 80 μM MG115, 80 μM MG132, 10 μM pepstatin A and one complete protease inhibitor cocktail tablet (Roche, USA)). After lysate centrifugation, supernatants were recovered as total proteins or chloroplast stroma proteins. Inhibition of proteasome-dependent degradation was accomplished by 40 μM MG115.

To isolate nuclear proteins from tobacco leaves, nuclei were first isolated from leaves, after which nuclear proteins were isolated using a plant nuclei isolation/extraction kit CelLytic^TM PN (Sigma-Aldrich, USA). According the manufacturer’s protocol, nuclei were collected from leaves with nuclei isolation buffer by mesh filtering. Cell lysate was prepared with 2.3 M sucrose by centrifugation at 12,000 × g for 10 min, after which the supernatant was discarded. The nuclei pellet was then added to nuclear protein extraction buffer. Nuclei proteins were then added to Working Extraction Buffer in addition with 80 μM MG115 and 80 μM MG132 and then centrifuged for 10 min at 12,000 × g. Pure supernatant was used to obtain nuclear proteins.

To extract cytosolic proteins from tobacco leaves, fresh leaves were homogenized with homogenization buffer (50 mM HEPES-KOH, pH 7.5, 250 mM sorbitol, 50 mM potassium acetate, 2 mM magnesium acetate, 1 mM EDTA, 1 mM EGTA, and 1 mM DTT, 80 μM MG115, 80 μM MG132, and one complete protease inhibitor cocktail tablet (Roche, USA)). After removal of cell debris by centrifugation for 10 min at 500 × g, sequential centrifugations of the supernatant at higher speeds were performed. Crude nuclear and chloroplast fractions were removed at 1,000 × g for 15 min; mitochondrial fraction was removed at 20,000 × g for 15 min; final supernatant was harvested to obtain the cytosolic proteins.

Proteins (100 μg of total proteins, 20 μg of chloroplast stroma proteins, 10 μg or 20 μg of nuclear proteins, or 20 μg of leaf cytosolic proteins) were separated by 4–12% Bis-Tris Plus (Novex, USA). Western blotting was performed using a Mini gel tank and Mini Blot Module (Life Technologies, USA) according to the manufacturer’s protocol. Proteins were transferred onto iBlot 2 NC Regular Stacks (Novex, Israel), after which blots were blocked using iBind Cards (Novex, Israel) according to the manufacturer’s instructions. NPR1-GFP proteins were detected by reacting with mouse monoclonal anti-GFP monoclonal antibody (Clontech, USA) and horseradish peroxidase-conjugated secondary antibody (Santa Cruz, USA). Bands were visualized using SuperSingnal West Substrate Working Solution (Thermo Scientific, USA) on X-ray film. To identify TOC proteins in the chloroplast outer envelope membrane, polyclonal rabbit antibody of Toc75–3 (Pisum sativum Toc 75 POTRA domain 3) (Agrisera, Sweden) was used. We employed primary antibodies for RubisCO protein, ubiquitin, and cellular proteins. Other primary antibodies were as follows: anti-RbcL (Agrisera), anti-His3 (Agrisera), and anti-actin (Agrisera).