Supersignal west femto maximum sensitivity western blot chemiluminescence substrate

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.

Dulbecco’s Modified Eagle’s Medium (DMEM), fetal calf serum (FCS), methionine-free DMEM, streptomycin, penicillin, protease inhibitors, trypsin, trypsin-EDTA, DEAE-dextran, protein A-sepharose, and triton X-100 were purchased from Sigma-Aldrich. Tissue culture dishes were purchased from Sarstedt (Nuembrecht, Germany). [³⁵S]-methionine was obtained from PerkinElmer (Waltham, Massachusetts). Acrylamide, Tris, TEMED, SDS, dithiothreitol (DTT), paraformaldehyde, polyvinyl difluoride (PVDF) membrane, ProLong Gold Antifade mountant with DAPI by Invitrogen, and sucrose were purchased from Carl Roth GmbH (Karlsruhe, Germany). Lubrol WX was obtained from MP Biomedicals (Eschwege, Germany). Endo-β-N-acetylglucosaminidase H (endo H) was acquired from Roche Diagnostics (Mannheim, Germany). TRIzol reagent (Life Technologies, California, USA), restriction enzymes, Isis proofreading DNA polymerase, molecular weight standards for SDS-PAGE, and SuperSignal™ West Femto maximum sensitivity western blot chemiluminescence substrate were purchased from Thermo Fisher (Schwerte, Germany).