Translation of Angiotensin-Converting Enzyme 2 upon Liver- and Lung-Targeted Delivery of Optimized Chemically Modified mRNA

Schrom, Eva; Huber, Maja; Aneja, Manish; Dohmen, Christian; Emrich, Daniela; Geiger, Johannes; Hasenpusch, Günther; Herrmann-Janson, Annika; Kretzschmann, Verena; Mykhailyk, Olga; Pasewald, Tamara; Oak, Prajakta; Hilgendorff, Anne; Wohlleber, Dirk; Hoymann, Heinz-Gerd; Schaudien, Dirk; Plank, Christian; Rudolph, Carsten; Kubisch-Dohmen, Rebekka

For in vitro experiments, cells were lysed with 0.5% Triton X-100 in ACE2 lysis buffer (1 M NaCl, 0.5 mM ZnCl 2 and 75 mM Tris-HCl, pH 7.5), while ex vivo samples were lysed with RIPA buffer (50 mM Tris, pH 8.0, 150 mmol/L NaCl, 1.0% Triton X-100, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate). Total protein content was determined by the bicinchoninic acid (BCA) assay, following the manufacturer's instructions (Thermo Fisher Scientific). If applicable, parts of the lysates were used for deglycosylation with NEB Protein Deglycosylation Mix II following the manufacturer's instructions for denaturing conditions (New England Biolabs). Cell lysates were separated on 8% or 4%-12% polyacrylamide gels (Thermo Fisher Scientific) and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad). Membranes were blocked in gelatin buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Triton X-100, 5 mM EDTA, and 0.25% gelatin) and probed with antibodies against ACE2 (0.1 mg/mL, AF933; R&D Systems/Bio-Techne) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10,000, 5174; Cell Signaling Technology/New England Biolabs). For protein detection, horseradish peroxidase (HRP)-conjugated anti-goat (1:10,000, sc2020) and anti-rabbit (1:10,000, sc2004; both from Santa Cruz Biotechnology) antibodies were added. For signal detection, Luminata Western HRP substrate was applied according to the manufacturer's protocol (Merck Chemicals).