Ab87436

The confluent A7r5 cells were placed in a quiescing medium (the same as that of the growth medium but with 1% FBS). A7r5 cells were treated with 2.5 mg/mL of SPHs for 24 h for ACE2 detection. EA.hy926 cells were treated with 2.5 mg/mL of SPHs for 18 h prior to the addition of 10 ng/mL of tumor necrosis factor-alpha (TNFα) for a 6 h co-treatment for detection of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). The dose of hydrolysate was selected based on our previous studies [9 (link)]. Cells were lysed in boiling Laemmle’s buffer containing 50 mM Dithiothreitol (DTT) and 0.2% Triton-X-100.
Cell lysates were loaded onto a 9% separating gel and transferred to a nitrocellulose membrane (diameter 0.45 µm, 1620115, Bio-Rad, Montreal, QC, Canada) for incubation with antibodies. Bands of ACE2 (ab87436, Abcam, Toronto, ON, Canada), ICAM-1 (sc-8439, Santa Cruz, Dallas, TX, USA) and VCAM-1 (sc-8304, Santa Cruz, Dallas, TX, USA) were normalized to α-tubulin (ab15246, Abcam). Donkey-anti-rabbit 800 CW or donkey-anti-mouse IRDye 680 RD secondary antibodies (Licor Biosciences, Lincoln, NE, USA) were used to visualize the fluorescent bands in a Licor Odyssey BioImager, which were quantified using Image Studio Lite 5.2 (Licor Biosciences).

Prior to the infection of the lung alveolar cell model, the culture medium was replaced with a control medium. In total, 200-nM SARS-CoV-2 spike protein (ACROBiosystems, SPN-C52H4) was added to the apical chamber of the Transwell insert for 3 h to confirm the cell binding ability with the SARS-CoV-2 spike protein. Subsequently, the cells were collected for western blotting, with the signals being detected by an Amersham™ Imager 600 system (GE Healthcare). For viral infection experiments, the SARS-CoV-2 spike-pseudotyped lentivirus (enhanced green fluorescent protein (eGFP) or luciferase reporter) was purchased from the National RNAi Core Facility of Academia Sinica, Taiwan; its RNA sequence was determined. The pseudoviruses were added to the apical chamber of the Transwell insert at the indicated MOI for 1 day. The pseudoviruses were subsequently removed and the cells were cultured in the control medium under the ALI condition. Three days after infection, a GFP signal was observed in the infected alveolar model. The cells were then stained with specific antibodies and the proinflammatory cytokine response was analyzed. For the neutralization assay, the lung alveolar model was pretreated with 20-μg/ml anti-ACE2 antibodies (Abcam, ab87436) for 30 min before infection for blocking the ACE2 receptors on the cell surface.