Fig. 3: Short ACE2 protein is expressed and not enriched on motile cilia relative to long ACE2 protein.
a, Schematic illustration of predicted long and short ACE2 protein isoforms and positions of antigen sequences used to generate the antibodies used; aa, amino acid. b, Representative western blots (n = 3 independent experiments) of Vero E6, HEK293, Caco2, RPE1, H441, 16HBE and BCi-NS1.1 cell line lysates immunoblotted with the anti-ACE2 CTD. c, Representative western blot of Vero E6, in vitro differentiated primary NEC and PBEC lysates, immunoblotted with anti-ACE2 CTD preadsorbed with the immunizing (blocking) peptide (right; n = 3 independent experiments) or similar charged control peptide (left; n = 1 independent experiment). d, Representative western blots (n = 3 independent experiments) of Vero E6 cells and in vitro differentiated nasal and bronchial cell lysates incubated with or without PNGase F, blotted with anti-ACE2 CTD (left panel) and anti-ACE2 NTD (right panel) antibodies. e, Representative immunofluorescence confocal images (n = 4 independent experiments) of in vitro differentiated PBECs stained with anti-α-tubulin (red), Alexa Fluor 555 phalloidin (blue), DAPI (gray) and ACE2 (green), detected with anti-ACE2 ECTO (top panel; antibody-detecting epitopes across the protein) or anti-ACE2 CTD (bottom panel). f, Schematic illustration of deciliation protocol using calcium shock (left) and western blot of whole and deciliated BCi-NS1.1 cells, deciliation wash and cilia pellet (right). g, Box and whisker plot of semiquantitative analysis of the western blots by densitometric analysis (n = 4 independent experiments). b–d and f, Gray arrow points to glycosylated long ACE2, black arrow to unglycosylated long ACE2 and red arrow to short ACE2.
