Radioimmunoprecipitation assay lysis buffer

Lysate samples were isolated from Calu-3 cells using a radioimmunoprecipitation assay lysis buffer (Wako Pure Chemical Industries, Osaka, Japan). Total protein was fractionated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane. After blocking with 5% bovine serum albumin in Tris-buffered saline/0.1% Tween-20 (TBST) at room temperature for 1 h, the membrane was incubated overnight at 4 °C with antibodies against ACE2 (1:1000; R&D Systems, Inc. Minneapolis, MN, USA) and GAPDH (1:2000, Santa Cruz Biotechnology, CA, USA). The membrane was washed three times with TBST and incubated with horseradish peroxidase anti-goat or anti-mouse secondary antibody (DakoCytomation, CA, USA) at 20 °C for 1 h. After washing three times with TBST, immunoreactive bands were detected using a ChemiDoc XRS+ Image System (Bio-Rad Laboratories, CA, USA). Protein bands were quantified using Image Lab software (Bio-Rad Laboratories).