Western Blot Analysis of Cellular Proteins

The treated cellular protein extracts were prepared as described previously (Lin et al., 2017 (link); Lee et al., 2018 (link)). In brief, equal amounts of protein were separated on an 8–10% SDS-PAGE gel and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dried milk for 30 min and then incubated with primary antibodies for 6–12 h at room temperature. The following primary antibodies were used: anti-ACE2 (1:1,000; Cell Signaling), anti-STAT3 (1:1,000; Cell Signaling), anti-TMPRSS2 (1:1,000; Abcam), anti-β-actin (1:10,000; Santa Cruz), and anti-GAPDH (1:10,000; Santa Cruz) antibodies. All the primary and secondary antibodies were diluted with 1% nonfat dried milk in Tris-buffered saline with 0.1% Tween 20 detergent. The membranes were washed using 0.1% Tris-buffered saline with Tween-20 and incubated in horseradish peroxidase–conjugated secondary anti-mouse or anti-rabbit antibodies (Santa Cruz, ratio: 1:5,000) for 1 h at room temperature. The membranes were washed for 1 h at room temperature. Chemiluminescent protein signals were detected by applying the SuperSignal West Pico PLUS chemiluminescent substrate (Pierce, catalog number: 34087).