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. Author manuscript; available in PMC: 2021 Aug 20.
Published in final edited form as: Eur Respir J. 2021 Jul 1;58(1):2003988. doi: 10.1183/13993003.03988-2020

FIGURE 3.

FIGURE 3

Effect of influenza A virus (IAV) on angiotensin-converting enzyme (ACE)2 protein levels and shedding in small airway epithelial cells (SAECs). a) Validation of ACE2 antibody (#AF933) using immunoblotting of recombinant human (rh)ACE2 protein and whole-cell lysate (1.6 μg) of human embryonic kidney (HEK)293T cells induced to overexpress human ACE2 (HEKACE2). b,c) Intracellular ACE2 protein in SAECs infected at the air—liquid interface with IAV (+) (H1N1 pdm09 virus, 3×105 pfu·transwell−1, 48 h) compared to uninfected cells (−), with indicated pre-exposure (+) to e-cigarette vapour (e-cig); b) ACE2 was detected by immunoblotting with the polyclonal antibody #AF933 and c) quantified by densitometry after normalisation to actin levels used as loading control. Cell lysates obtained from distinct donors (#1–3) are noted. d,e) Released ACE2 protein in apical supernatants (normalised by volume) of SAECs infected with IAV (+) at the air—liquid interface, with the indicated pre-exposure (+) to e-cig; ACE2 d) detected by immunoblotting with #AF933 antibody and e) quantified by densitometry. Supernatants obtained from cells from distinct donors (#1–4) are noted. f) Released ACE2 protein in apical supernatants from SAECs infected while submerged in culture media with lower dose (LD; 0.5×105 pfu·transwell−1) or higher dose (HD; 1.0×105 pfu·transwell−1) IAV for 48 h; ACE2 was detected by immunoblotting (with antibody # 21115-1-ap). Cell lysate (1.6 μg) from HEKACE2 was used as control. Graphs show individual data points from independent experiments, mean±SEM; t-test.