Anti ace2

Cell lysates were collected with RIPA lysis buffer and quantified using a micro-BCA kit (Thermo Fisher Cat#: 10249133). Samples were boiled for 10 min at 95 °C in 5x Laemmli buffer, separated in an SDS-PAGE gel, and transferred to a polyvinylidene difluoride membrane. Membranes were incubated with 5% non-fat milk in tris-buffered saline buffer (10 mM Tris, pH 8.0, 150 mM NaCl) with 0.1% Tween-20 detergent (TBST) for 1 h and incubated with anti-ACE2 (Bioss Inc Cat #: BS-1004R), anti-NRP1 (Bio-Techne Corporation Cat#: NBP2-67539), anti-TMPRSS2 (Bio-Techne Corporation Cat#: NBP3-00492), anti-Phospho-AMPKα (Thr172) (Cell Signaling Cat#: 2531), and anti-β-actin (Sigma Aldrich Cat#: A5441) primary antibodies at 4 °C for 16 h (dilution 1:1000 or 1:500 for the pull-down assay). Membranes were washed thrice for 10 min and incubated with a secondary anti-rabbit-HRP antibody (Cell signaling Cat#:7074) for 1 h (1:5000 dilution). Blots were washed again with TBST and developed with ECL substrate (Bio-Rad Cat#: 1705061) on an iBright system (Invitrogen). Band densitometry was performed using ImageJ.

Whole-cell extracts were prepared using ProteoJET Mammalian Cell Lysis Reagent (Fermentas, Burlington, Canada) supplemented with protease and phosphatase inhibitors (Fermentas) according to the manufacturer’s instructions. Protein (20–40 mg) was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis in 8%–10% gels and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA). The blots were blocked for 1 h at room temperature with 5% bovine serum albumin (Sigma) in Tris-buffered saline/0.1% Tween-20. Next, the blots were probed with anti-ACE2 (Bioss, Beijing, China) or anti-GAPDH (Proteintech, Wuhan, China) antibodies overnight at 4°C. The blots were then incubated with corresponding horseradish peroxidase- conjugated GAPDH (Zsbio, Beijing) for 1 h at room temperature. After additional washes, signals were detected using SuperSignal ECL (Pierce, Rockford, IL).