Protease inhibitor cocktail without edta

4 mg mitochondria from NTC- or shTFB1M-expressing PLC cells treated with 100μg/ mL Chloramphenicol & Thiamphenicol for 30 min was lysed with lysis buffer (260 mM sucrose, 100 mM KCl, 20 mM MgCl₂, 10 mM Tris–Cl [pH 7.5], 1% Triton X-100, 5 mM β-mercaptoethanol, protease inhibitor cocktail without EDTA (Roche), 10 U/μl RNase inhibitor(BBI), 100 μg/ml Chloramphenicol & Thiamphenicol) on ice for 30 min. Lysate was precleared by centrifugation at 10 000 × g for 15 min at 4°C, then the lysates were loaded on a 4 ml 10–30% continuous sucrose gradient (10 mM Tris–Cl, 100 mM KCl, 20 mM MgCl_2, 5 mM β-mercaptoethanol, 10 μg/ml Chloramphenicol & Thiamphenicol, protease inhibitor cocktail without EDTA, 1 U/μl RNase inhibitor) and centrifuged at 180 000 ×g for 4 h in a Beckman MLS-50 rotor at 4°C. After centrifugation, gradients were fractionated from the top into 24 equal fractions. Half of each fraction was used for RNA isolation by TRIzol Reagent (Ambion). Isolated RNA was dissolved in 100 μl DEPC water and the RNA concentration was detected by spectrophotometer (One Drop). The other half of each fraction was mixed with adjacent half fraction together and then was subjected to western blot analysis using the anti-MRPS15, anti-MRPS16, anti-MRPS34, anti-MRPL13, anti-MRPL48 antibodies.

Aha1 I64X (2YA6,64C1) was expressed and crosslink products were formed by irradiation with UV light. Disruption was carried out using a Mixer Mill MM 200 (Retsch) with a shaking frequency of 30 s⁻¹. The resulting cell powder was resuspended in aqueous buffer without detergent (50 mM Tris-Cl pH 7.5, 500 mM NaCl, 1 mM PMSF, 1× protease inhibitor cocktail without EDTA (Roche)). Soluble proteins were separated from the insoluble proteins by a 100,000× g centrifugation for 30 minutes. Insoluble proteins were solved in an insoluble protein buffer (50 mM Tris-Cl pH 7.5, 8 M urea, 2% SDS, 150 mM NaCl, 2 mM DTT, 1 mM PMSF, 1× protease inhibitor cocktail without EDTA (Roche)).
For translational inhibition assays using cycloheximide Aha1 I64X (2YA6,64C1) was expressed and cycloheximide (final concentration 50 µg/ml) was added to the culture prior to UV-irradiation for different periods of time.

Angiotensin II-converting enzyme activity was measured by a fluorometric enzymatic assay, using an artificial substrate of the enzyme, hippuryl-L-histidyl-L-leucine (Sigma-Aldrich). The hippuryl-L-histidyl-L-leucine substrate was prepared at 25 mM in 25 mM NaOH solution. Lung and kidney samples were homogenized in buffer containing 50 mM HEPES at pH 7.4, 25 mM ZnCl₂, 150 mM NaCl, Triton X-100 and 0.5% protease inhibitor cocktail without EDTA (Roche). Homogenates were centrifuged for 10 min at 14,000 g and 6°C to clarify. The protein concentration was measured in the supernatants of each sample, and 1 µg of protein was mixed with the substrate solution consisting of 5 mM hippuryl-L-histidyl-L-leucine, 0.9 M NaCl and 0.4 M borate buffer at pH 8.3 to a final volume of 75 μl. The samples were incubated at 37°C for 20 min, and the reaction was stopped by adding 180 μl of 0.28 M NaOH. Subsequently, 15 μl of o-phthaldialdehyde (20 mg/ml in methanol) was added (Sigma-Aldrich), whose reaction product emitted fluorescence. After 10 min at room temperature, the reaction was acidified with 30 μl of 3 N HCl. Each sample was centrifuged for 5 min at 800 g, and 200 μl of the supernatant was transferred to a black 96-well plate. The fluorescence was measured at an excitation wavelength of 360 nm with emission at 485 nm.

Fresh, independent colonies of Chlamydomonas reinhardtii cells (CC-1883) were cultured and maintained as previously described^{1 (link)}. For IP, lysates were prepared by harvesting cells in mid-log phase (OD₇₅₀~0.6). Cell cultures were incubated on ice for 30 min prior to centrifugation at 4000 rpm, 5 min, 4 °C. The pellet was re-suspended in IP lysis buffer (1 × PBS, NaF 50 mM, NaSVO₄ 1 mM, MgCl₂ 5 mM, PMSF 1 mM, NP40 0.1%, DTT 5 mM, NaCl 0.8 M, 1X protease inhibitor cocktail without EDTA (Roche)). The resuspended cells were then dounce homogenised on ice 20×, followed by centrifugation at 4700 rpm, 4 °C, 30 min. The clarified samples were either snap frozen in liquid nitrogen or 10% TCA-acetone precipitated and resuspended in resuspension buffer (Urea 8 M, NaCl 0.5 M and DTT 5 mM) for western blot analysis.

DNA sequences encoding His₆ (6 × histidine) and 4 × HA were inserted upstream of Arabidopsis SUMO1 cDNA, and the resulting recombinant His₆‐HA₄‐SUMO1 DNA was introduced into the β‐estradiol‐inducible vector pER8 61. The construct was transformed into Arabidopsis by the floral dip method 60 to generate a SUMO1‐overexpressing Arabidopsis line. SUMO1 conjugates were assessed in plants carrying the XVE‐His₆‐HA₄‐SUMO1 transgene. Plants were grown on MS media for 2 weeks before being treated with 10 μm β‐estradiol for 15 h under light conditions and then being directly exposed to air for 4 h. Samples were harvested, ground in liquid nitrogen, and resuspended in extraction buffer (20 mm Tris/HCl pH 8.0, 8 m urea, 100 mm NaH₂PO₄, 1% Triton X‐100, 10 mm β‐mercaptoethanol) containing 1× protease inhibitor cocktail without EDTA (Roche, Basel, Basel‐Stad, Switzerland) and 20 mm imidazole (Sigma). After centrifugation, supernatants were purified on Ni²⁺‐NTA columns using a 20–500 mm imidazole concentration gradient, according to the manufacturer's instructions (Qiagen, Hilden, North Rhine‐Westphalia, Germany). Eluted proteins were detected by western blot analysis with an anti‐HA antibody (Santa Cruz Biotechnology) or an anti‐AtSIZ1 antibody 33.