Pa5 20812

Cells were placed in quiescing media (containing 1% FBS) until reaching confluency. VSMCs were treated with peptides (50 µM) for 24 h for the detection of AT₁R and MasR; ECs were treated with peptides (50 µM) for 18 h for the detection of TNFα receptors 1/2 (TNF-R1/R2). Peptide concentrations and time of treatment were selected per our previous studies [28 (link),33 (link)]. After the treatment, cells were scraped and lysed in a boiling Laemmle’s buffer with 50 mM DTT and 0.2% Triton-X-100.
Cell samples were run in a 9% separating gel and transferred to a nitrocellulose membrane before being incubated with specific primary antibodies. Protein bands of AT₁R (PA5-20812, Invitrogen), MasR (NBP1-78444, Novus Biologicals, Toronto, ON, Canada), TNF-R1 (sc-8436, Santa Cruz, Dallas, TX, USA), TNF-R2 (sc-8041, Santa Cruz), glutathione peroxidase 4 (GPx4; ab125066, Abcam, Toronto, ON, Canada), and superoxide dismutase 2 (SOD2; ab227091, Abcam) were normalized to α-tubulin (ab15246, Abcam) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH, ab8245, Abcam). The fluorescent bands were visualized by adding donkey-anti-mouse IRDye 680 RD or donkey-anti-rabbit 800 CW secondary antibodies (Licor Biosciences, Lincoln, NE, USA), and the signals were detected using Licor Odyssey BioImager (Licor Biosciences).

Heat induced antigen retrieval (HIAR) was performed for 10 min with citrate buffer (pH6.0) and cooled for a further 15–20 min with agitation to room temperature. Endogenous peroxidase was blocked with 2% hydrogen peroxide for 30 min followed by washing with Dako EnV Flex wash buffer (# K800721-2, Dako, CA) and a further background block was performed with SNIPER (# BC-BS966H, Biocare Medical, CA) for 30 min. Sections were buffer washed and followed by a final protein block for 30 min. A buffered wash followed by incubation in the polyclonal primary antibodies; AT1R (1 µg/ml, # PA5-20812, Invitrogen, MA), AT2R (2 µg/ml, # PA5-20813, Invitrogen, MA) and AT4R (1:200, # PA5-23777 [LNPEP], Invitrogen, MA), overnight in a humidity chamber at 4 °C. Antibody diluent (Dako REAL antibody diluent – # S202230-2, Dako, CA) was substituted for primary antibodies as method controls. A Rabbit specific HRP/DAB Micro-polymer IHC Detection Kit (# ab236469, Abcam, Bristol, United Kingdom) was used according to manufacturer’s instruction, with Goat anti-rabbit HRP conjugate serving as the secondary antibody. Visualization of the antibody’s adherent sites was achieved with chromogen 3.3′-Diaminobenzidine (DAB). Nuclei was counterstained with Shandon Haematoxylin (# 6765015, Thermo Scientific, MI) followed by dehydration, clearing and mounting in dibutylphthalate polystyrene Xylene (DPX).

SARS-CoV-2 infected AEC transwell inserts were fixed at room temperature for 30 min by adding 600 µL of 10% formalin to the basolateral chamber and 200 µL of 10% formalin to the apical surface of AEC transwells. Both apical and basolateral surfaces of transwell inserts were washed 3 times with PBS. The tissue was treated for 30 min in blocking solution (5% FBS/0.05% Saponin in PBS) in the apical chamber. AECs were then incubated with primary antibodies for ACE2 (1:100; AF933, R&D Systems) and ATR1 (1:500; PA5-20812, Thermofisher) in blocking solution for 90 min at room temperature. Tissues were washed three times in PBS and then incubated with secondary antibodies (1:500; Donkey anti-Goat Alexafluor 488 and Donkey anti-Rabbit Alexafluor 568, Thermofisher) in blocking solution for 60 min at room temperature. Tissues were again washed 3 times in PBS. Transwell membranes were then carefully cut using an 8 mm biopsy punch and mounted to glass slides with mounting media (Vectashield). Images were acquired using confocal microscopy (TCS SP5, Leica).