Quantitative Analysis of ACE2 Protein

Tissue samples were mechanically disrupted by homogenisation using a bead-beating tissue homogeniser after adding lysis buffer (50 mM Hepes, pH 7.4, 150 mM NaCl, 10% glycerol, 1% NP-40, 1 mM CaCl₂) containing protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Immunoblotting was carried out as formerly described [52 (link)]. Briefly, 100 µg of each sample was separated on an 8% SDS–PAGE gel. The reaction was performed using antibodies as indicated in Supplementary Material Table S2. In advance, tissue homogenate derived from the kidney was used as a positive control. Protein bands were visualised using SuperSignal™ West Femto maximum sensitivity Western blot chemiluminescence substrate (Thermo Fisher Scientific, Waltham, MA, USA) and a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). ACE2 band intensities were quantified via ImageJ v8 and normalised against β-actin.