Foxo1

For Western blotting, cells and tissues were lysed in RIPA buffer (Tris-HCl 25 mM, pH 7.6; NaCl 150 mM; NP-40 1%; sodium deoxycholate 1%; SDS 0.1%) with protease inhibitors and phosphatase inhibitors. Protein concentration was measured using bicinchoninic acid (BCA) (ComWin Biotech, China). Equal amounts of protein were separated by SDS-PAGE and transferred to a PVDF membrane which was incubated in 5% milk in Tris-buffered saline for 1 h at room temperature, followed by incubation with primary antibodies against ACE2 (Cell Signaling Technology, USA, 1:1000 dilution), FoxO1 (Cell Signaling Technology, USA, 1:1000 dilution), SARS-CoV-2 Nucleocapsid (NP) (SinoBiologicals, China, 1:1000 dilution), HA (Cell Signaling Technology, USA, 1:1000 dilution) and Flag (Cell Signaling Technology, USA, 1:1000 dilution) overnight at 4°C, and a secondary antibody (1:10000 dilution) conjugated with horseradish peroxidase (Cell Signaling Technology, USA) for 1 h at room temperature. Membranes were developed with chemiluminescent ECL reagents (Millipore, USA). The relative expression of target protein to the control β-actin were determined by densitometric analysis using ImageJ software (National Institutes of Health, USA).