Protease inhibitor cocktail p9599

A. tumefaciens was grown overnight in LB medium containing the appropriate antibiotics, collected by centrifugation, and then suspended in 10 mM MgCl₂ containing 100 mM acetosyringone. After incubation at RT for at least 2 hr, the cultures were diluted to an OD₆₀₀ = 0.5. Leaves of four‐week‐old N. benthamiana plants were agroinfiltrated using a needleless syringe and plants were returned to the greenhouse for 72 hr. Samples from agroinfiltrated leaves were lysed in a buffer containing 50 mM Tris (PH 7.6), 150 mM NaCl, 0.5% Triton X‐100 and protease inhibitor cocktail P9599 (Sigma, MO). Extracts were centrifuged at 14,000 × g for 15 min at 4°C. Anti‐Myc or anti‐GFP traps (Chromotek) were used for co‐immunoprecipitation experiments according to the manufacturer's instructions. Immunoblot analysis was performed as previously described (Willmann et al., 2011 ), using anti‐myc or anti‐GFP antibodies (Sigma‐Aldrich) at a dilution of 1:3000.

Leaves were placed into mortar containing liquid nitrogen and pulverized with a pestle immediately after treatments. 0.4 g of leaf powder was resuspended with 2 mL of ice-cold 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 1 mM EDTA, 10 mM NaF, 2 mM sodium orthovanadate, 1 mM sodium molybdate, 10% (v/v) glycerol, 0.1% Tween 20, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 1× protease inhibitor cocktail P9599 (Sigma-Aldrich, St. Louis, MO, USA). Than the samples were Ultra-Turrax (IKA, Staufen, Germany) treated for 15 s and centrifuged a 12,000× g for 10 min at 4 °C. The supernatant thus obtained was mixed with protamine sulfate (PS) at a final concentration of 0.05% to partially remove the RuBisCO enzyme [65 (link)]. The sample was maintain on ice for 30 min and centrifuged at 12,000× g for 10 min at 4 °C. Proteins in the supernatant were than subjected to methanol/chloroform precipitation. Briefly sample was added by methanol/chloroform/water (4:1:3 v/v), agitated vigorously and centrifuged for 10 min at 10,000× g at 4 °C. The upper layer was then removed without disturbing the interface and sample was further added of three volumes of methanol and centrifuged. The final pellet was dried, dissolved in 8M Urea, 4% (w/v) CHAPS, and 20mM DTT, and subjected to 2DE.

Protein was extracted from each tissue using extraction buffer [50 mM Tris-MES pH 7.5, 50 mM NaCl, 0.5% SDS (v/v)], and protease inhibitor cocktail P9599 (Sigma-Aldrich, St. Louise, MO, United States). The extract was centrifuged at 12000 rpm at 4°C for 30 min. For immunoblotting, protein samples (18 μg total protein) were separated on 12% SDS PAGE gels and transferred to nitrocellulose membranes (BioTrace^TM NT nitrocellulose, Mexico) followed by western blot analysis. After blocking non-specific binding with 5% milk, the blot was subsequently incubated with antibodies generated against the indicated proteins and detected using the Super Signal^TM West Pico PLUS Chemiluminescent Substrate kit (Thermo Scientific, United States).

Plant material was extracted by grinding precooled samples in liquid nitrogen with a pestle to a fine powder^{44 (link)}. The extraction buffer (50 mM Tris–HCl, pH 7, and 1% SDS with Sigma P9599 protease inhibitor cocktail) was added, vortexed vigorously, and then centrifuged for 30 min at 14,000 rpm to obtain a supernatant. Protein content in the supernatant was quantified with Bio-Rad DC reagent (Lowry method). Samples were dissolved in Laemmli buffer, treated at 95 °C for 10 min, and loaded (40 mg) onto Invitrogen NuPAGE gels (10%Bis–Tris Midi Gels). After electrophoresis, proteins were transferred to a PVDF membrane using the Bio-Rad Trans-Blot turbo transfer pack. An anti-ADH antibody (Agrisera AS10685, Agrisera, Vännäs, Sweden), an anti-PDC antibody (Agrisera AS10691; Agrisera, Vännäs, Sweden), and secondary goat anti-rabbit IgG HRP conjugated antibody (Agrisera AS09 602; Agrisera, Vännäs, Sweden) were used. Detection was performed using the LiteAblot TurboChemiluminescence substrate (Euroclone). amido black staining was performed to check equal loading. The blot was stained for 10 min [0.1% amido black (Sigma Aldrich, Milan, Italy), 45% methanol, 10%acetic acid] and then washed in a destaining solution (90%methanol/2% acetic acid/8% water) for 2 min.

N. benthamiana leaves or Arabidopsis seedlings were ground to a fine powder in liquid nitrogen with sand (Sigma-Aldrich). Proteins were extracted in buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 10 mM DTT, 1 mM NaF, 1 mM Na2MoO4.2H2O, 1% Phosphatase Inhibitor Cocktails 2 and 3 (Sigma-Aldrich), 1% (v/v) P9599 Protease Inhibitor Cocktail (Sigma-Aldrich), 100 μM phenylmethylsulphonyl fluoride and 0.5% (v/v) IGEPAL CA-630 (Sigma-Aldrich). Extracts were incubated 30 min at 4°C and centrifuged for 20 min at 16,000 g at 4°C. Supernatants were incubated for 1–2 h at 4°C with GFP-Trap (ChromoTek) or ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich), and washed 3–4 times with extraction buffer containing 0.1–0.5% (v/v) IGEPAL CA-630 (Sigma-Aldrich). For GFP-Trap immunoprecipitation, beads were boiled in NuPAGE LDS sample buffer (Thermo Scientific) to release proteins. FLAG peptide was used for specific elution of anti-FLAG immunoprecipitated proteins. For immunoprecipitation in Arabidopsis protoplasts, protoplasts were transfected with indicated plasmids, incubated overnight and then treated with H₂O or 1 μM flg22 or elf18 for 15–30 min. Proteins were extracted with extraction buffer (50 mM HEPES [pH 7.5], 150 mM KCl, 1 mM EDTA, 0.5% Trition-X 100, 1 mM DTT, proteinase inhibitor cocktail) at 4°C. Immunoprecipitation was then carried out as described above.