Pefabloc sc plus

Kidney cortical tissue was prepared for immunoblot analysis with antibodies against phosphorylated and total Drp1. Kidney cortex samples were homogenized in extraction buffer containing 50 mM HEPES, pH7.4, 150 mM NaCl, 0.5% Triton X-100, 0.025 mM ZnCl2, 0.1 mM Pefabloc SC Plus (Roche, Basel, Switzerland), EDTA-free protease inhibitor cocktail tablet (Roche, Basel, Switzerland), and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Protein concentration was determined using the Micro BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA).
Western blot was performed by separating 15 µg of total protein in 7% SDS-polyacrylamide gels and transferring it onto PVDF membranes (Immobilon-P Millipore, Burlington, MA, USA). Membranes were incubated in skimmed milk blocking solution (5%) for 1 h and incubated overnight at 4 °C with galectin-3 (1:500; 126701, Biolegend, San Diego, CA, USA) in 2.5% skimmed milk. HRP-conjugated anti-mouse IgG antibody was used as secondary antibody. β-Actin antibody (1:20,000; BS1003, Bioworld, Dublin, Ireland) was used as loading control.
Proteins were detected in films (AGFA CURIX, Mortsel, Belgium) after 3-min incubation with Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA). Protein bands were quantified by densitometry with the ImageJ software.

Kidney cortex samples were homogenized in a buffer consisting of 50 mM HEPES, pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 0.025 mM ZnCl₂, and 0.1 mM Pefabloc SC Plus (Roche) and EDTA-free protease inhibitor cocktail tablet (Roche) and clarified by centrifugation at 14,000×g for 10 min at 4°C. After measuring protein concentration by Micro BCA assay kit (Thermo Scientific), tissue samples were diluted in a buffer containing 100 mM Tris-HCl, 600 mM NaCl, 10 µM ZnCl2, pH 7.5 and 100 µM captopril, 5 µM amastatin, 5 µM bestatin (all from Sigma-Aldrich), and 10 µM Z-Pro-prolinal (Enzo Life Sciences). To each well, 40 µl of a diluted tissue sample (0.5 µg of total renal protein) was added, along with 10 µl of buffer (with or without an specific ACE2 inhibitor, MLN-4760), and the reaction was initiated by the addition of 50 µl of the substrate (5 µmol/l, final concentration). Kidney cortex ACE2 activity was determined after 4-hour incubation at 37°C. The plates were read as described above. Experiments were carried out in duplicate for each data point. Results after subtraction of the inhibition value were expressed as RFU (Relative Fluorescent Units) per µg of protein and per hour (RFU/µg/hr).