Complete protein inhibitor cocktail

Mitochondria were isolated from cultured cells using mitochondria isolation kit (Abcam, ab110168). Cells were harvested and ruptured with a Dounce homogenizer in the buffer A containing cOmplete protein inhibitor cocktail (Roche) and 1 mM phenylmethylsulfonyl fluoride (PMSF). 15 stokes were performed with a motorized pestle operated at 300 rpm. Nuclei and unbroken cells were removed by centrifugation at 600 × g. Homogenates were spun at 7000 × g for 15 min to precipitate mitochondria. Mitochondria pellets were collected for downstream uses. To isolate mitochondria from zebrafish, skeletal muscles of fish were dissected and minced. Tissue was disrupted in muscle mitochondria isolation buffer (67 mM sucrose, 50 mM KCl, 1mM EDTA, 0.2% fatty acid free BSA, 50 mM Tri-HCl, pH7.4) with a Dounce homogenizer tight pestle operated at 1300 rpm. The extract was centrifuged at 600 × g for 5 min to clear intact myofibrils and heavy cell debris and then spun at 7000 × g for 15 min to isolate the desired mitochondrial fraction.

Vascular leakage was determined by extravasation of FITC-labeled dextrans. Fifteen minutes before killing the rats, 0.5 mL FITC-labeled dextrans of 70 kDa (12.5 mg/mL; FD70S-1G, Sigma-Aldrich; Saint Louis, Missouri, USA) were administered intravenously. Fifty mg lung tissue was homogenized in RIPA buffer with protease and phosphatase inhibitors (cOmplete™ Protein Inhibitor Cocktail, Roche) and centrifuged for 15 min at 13,000 rpm at 4 °C. Fluorescence intensity of lung homogenates was determined at an excitation wavelength of 485 nm and an emission wavelength of 528 nm using a spectrophotometer (SpectraMax® M2e) and converted to µg/mg tissue using a standard curve.

293T cells were transiently transfected with GFP-CDK4 plasmid. At 36 h after transfection, cells were harvested, and total cell lysates were extracted using cell lysis buffer (9803, Cell Signaling Technology, USA) containing a complete protein inhibitor cocktail (04693116001, Roche, Germany) on ice. The obtained lysates were incubated with rotation at 4°C for 30 min. After centrifugation, the supernatant (1000 μL) was divided into two aliquots: 450 μL (50 μL was removed, denatured, and stored to check the input by western blotting) was incubated with 2 µg anti-CDK4 antibody (sc-260); and 450 μL (50 μL was removed, denatured, and stored to check the input by western blotting) was incubated with a nonspecific antibody (rabbit IgG, sc-2749). After incubation overnight at 4°C, 30 µL protein A/G-coated magnetic beads (Thermo Fisher Scientific) were added to each sample and incubated for 2–4 h at 4°C. Extracts were sequentially washed five times with 1× cell lysis buffer using a magnetic stand. Finally, 60 µL protein elution buffer and 0.6 µL DTT were added, and samples were heated at 37°C for 2 h to denature and separate the magnetic beads. The samples without magnetic beads were subjected to western blotting analysis.

Cells were washed with ice-cold phosphate-buffered saline and lysed in a lysis buffer (50 mmol/l Tris-HCl, pH 8.0; 150 mmol/l NaCl; 1% NP-40) supplemented with complete protein inhibitor cocktail (Roche, Penzberg, Germany). Cell lysates were incubated with 2 μg rabbit anti-eIF3d antibody or mouse anti-GRK2 antibody overnight at 4 °C and precipitated with 20 μl protein G-Sepharose 4 Fast flow (Thermo, Waltham, MA, USA) for 4 h at 4 °C. The co-immunoprecipitates were washed four times with the lysis buffer and boiled for 5 min at 100 °C in protein loading buffer. Immunoprecipitated proteins were detected by following immunoblots.