Protease inhibitor cocktail s8830

Agrobacterium strain GV3101 carrying the targeted plasmid was resuspended to OD ₆₀₀ = 0.4 in an injection buffer (10 mM MES [pH 5.6], 10 mM MgCl₂, 150 μM Acetosyringone), then gently injected into the tobacco leaf using a 1-mL needle-free syringe. The tobacco was cultivated in the dark for 12 h, then transferred to the short-day growth conditions. For cell death leaf observation experiments, the pictures were taken at 3 or 4 days after injection. Then, the indicated N. tabacum leaves were stained with trypan blue and decolorized overnight. For protein extraction experiments, the samples were taken 36 h after injection (before cell death occurrence). The leaves were frozen quickly in liquid nitrogen and ground into powders in a mortar. Then, the sample was transferred to a new 2-mL centrifuge tube. Native extraction buffer (50 mM Tris-MES [pH 8.0], 0.5 M Sucrose, 1 mM MgCl₂, 10 mM EDTA, 5 mM DTT, and 1% [w/v] protease inhibitor cocktail S8830 [Sigma, St. Louis, MO, USA]) was added in a ratio of 1 g to 2 mL. The tubes were placed on ice for 30 min and centrifuged at 12,000 g for 15 min, and then the supernatant was transferred to a new centrifuge tube to be used in the next protein immunoblotting assay performed as described previously [59 (link)].

Protoplast proteins were processed in extraction buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 10% (v/v) glycerol, 2 mM EDTA, 5 mM DTT, 1% [w/v] protease inhibitor cocktail S8830 [Sigma], 0.1% Triton). Lysates were centrifuged for 15 min at 14,000 rpm at 4 °C. Aliquots of supernatants were used as input samples. For immunoprecipitation assays, 20 μL GFP-Trap Magnetic Agarose (Chromotek, Munich, Germany) was added to 1 mL total protein extract for 2 h incubation at 4 °C. Beads were collected by centrifugation and washed four times with a PBS buffer containing 0.1% (v/v) Triton X-100. Then, beads were resuspended in 80 μL of PBS buffer with SDS loading buffer and incubated at 95 °C for 8 min. Proteins were separated via SDS-PAGE gels and analyzed by immunoblotting. The anti-bodies used were anti-GFP (Sigma Aldrich, Louis, MO, USA, 11814460001) and anti-MYC (Sigma Aldrich, Louis, MO, USA, 11867423001).

Protein was extracted from soybean leaf tissues using extraction buffer (50 mM Tris-MES pH 8.0, 0.5 M sucrose, 1 mM MgCl₂, 10 mM EDTA, 5 mM DTT) with protease inhibitor cocktail S8830 (Sigma-Aldrich, St. Louise, MO, USA) added as described by [23 (link)]. The extract was centrifuged at 12,000 rpm at 4 °C for 30 min and the supernatant was collected. For immunoblotting, the extracted proteins were separated by SDS-PAGE (10% acrylamide gel) and transferred to PVDF membrane (Millipore, Billerica, MA, USA) by semi-dry electro-transfer (Bio-Rad, Hercules, CA, USA). The membrane was blocked in 1× TBST buffer containing 5% milk powder for 2 h. After washing, the membrane was further incubated with anti–Phospho-p44/p42 MAPK (anti-pTEpY) diluted at 1:3000 (Cell Signaling Technology, Danvers, MA, USA), followed by incubation with secondary antibody diluted at 1:7500. The bands were visualized by incubating with chemiluminescent HRP substrate (Millipore, Billerica, MA, USA).