Fig. 5: Stability of the ACE2 and MIRb-ACE2 translation products.
a, Flow cytometric detection of GFP expression (left) and quantification of mean frequency (± s.e.) of GFP-expressing cells (right) in HEK293T cells transfected to express either ACE2 or MIRb-ACE2 in conjunction with a FLAG tag and GFP, linked by a P2A peptide. Symbols represent three independently transfected cultures in the same experiment. One representative experiment of three is shown. b, Detection of ACE2 and putative MIRb-ACE2 protein by western blotting for the FLAG tag in cell lysates from the same cells as in a. Titration of the transfection plasmids used is also indicated. One representative experiment of two is shown. c, Detection of MIRb-ACE2 protein by western blotting for the FLAG tag in HEK293T cells transfected with increasing amounts of the expression plasmid (top) and mean (± s.e.) MIRb-ACE2 expression, determined by RT–qPCR in the same cells, in comparison with MIRb-ACE2 expression in IFN-α-stimulated NHBE cells and SCC-4 and SCC-25 cells (bottom). Each symbol represents the mean value of two technical RT–qPCR replicates of a single culture, and the bars and error bars represent the mean and s.e. of the three independently treated cultures in the same experiment. d, Detection of ACE2 and MIRb-ACE2 protein by western blotting for the FLAG tag in cell lysates from HEK293T cells transfected (with 4 µg of expression plasmids) to express either wild-type isoform or either isoform with the two lysine residues mutated (K2R; all in conjunction with a FLAG tag and GFP, linked by a P2A peptide). HEK293T cells transfected to express the wild-type isoforms were treated with the MG-132 inhibitor. One representative experiment of two is shown. e, Stability of ACE2 and MIRb-ACE2 protein, determined by western blotting in HEK293T cells transfected to express either isoform, after the indicated times following treatment with cycloheximide. Data from a single experiment are shown. f, Kinetics of mean (± s.d.) ACE2 enzymatic activity in the supernatant of HEK293T cells transfected to express either ACE2 or MIRb-ACE2 or both (ACE2 + MIRb-ACE2). Expression plasmids were used at 4 µg and 2 µg each for individual transfections and co-transfections, respectively. Symbols represent the mean value of two technical replicates in the same experiment. One representative experiment of two is shown. RLU, relative light units. g, Flow cytometric detection of SARS-CoV-2 S1 bindings to HEK293T cells transfected to express either ACE2 or MIRb-ACE2 or both (ACE2 + MIRb-ACE2). ACE2 and MIRb-ACE2 expression plasmids were used at 4 µg and 14 µg for individual transfections, respectively, and at 2 µg and 14 µg for co-transfections, respectively.
