Anti flag

Receptor-expressing cells were lysed with RIPA buffer (Sigma) and centrifuged for 15 min at 15,000 rpm and 4 °C, and the cell lysates were collected. Purified hFc-tagged S-ECD, RBD, NTD, or S2 domain proteins (final concentration of 10 µg/mL) were added to the cell lysate together with anti-FLAG beads (Sigma) or protein A beads (SMART Lifesciences), and the mixture was incubated at 4 °C overnight. The beads were washed three times with RIPA buffer, and the samples were prepared for western blot analysis with anti-hFc (ABclonal, AS002) or anti-FLAG (Smart Life Sciences, SLAB01) antibodies. For measurement of the Kd values, the receptor coding plasmid was cotransfected with the CFP reporter vector (5:1) into HEK293E cells. Cells were collected 48 h after transfection. Approximately 10⁴ cells per well were used for binding with a series of diluted purified S-ECD-hFc proteins as described in the receptor profiling experiment. The flow data were analyzed with FlowJo software. The degree of ligand binding at each ligand concentration was calculated by normalizing the mean APC fluorescence intensity (APC-MFI) of receptor-positive cells with that at a ligand concentration of zero. The Kd and Bmax (maximum binding) values were calculated using Prism8 software.