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. 2023 Jul 28;9:78. doi: 10.1038/s41421-023-00581-9

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a Western blotting analysis of cell lysates of HEK293 cells transiently over-expressing FLAG-tagged ACE2 orthologs from different bat species using anti-FLAG M2 antibodies. β-actin served as the loading controls. b Transduction of HEK293 cells expressing ACE2 orthologs from different bat species by SARS-CoV S, SARS-CoV-2 S, BANAL-20-52 S, and BANAL-20-236 S pseudovirions. The experiments were done in triplicate and repeated at least three times. One representative is shown with error bars indicating SD. c Alignment of RBD amino acid sequences from SARS-CoV-2-related sarbecoviruses. RBMs, receptor binding motifs, are indicated by dashed red line box. Residues 372, 493, and 498 are shaded in yellow background. d Detection of SARS-CoV-2 S, BANAL-20-52, or BANAL-20-236 S mutants in cell lysates and pseudovirions by western blot assay using anti-FLAG M2 antibodies. β-actin and gag-p24 served as loading controls (cell lysates, top panel; pseudovirions, bottom panel). e, f Transduction of 293 cells expressing human ACE2, R. malayanus ACE2, R. sinicus HB ACE2, or P. abramus ACE2 by WT and Q498H mutant SARS-CoV-2 S pseudovirions (e), and WT and H498Q mutant BANAL-20-52 S pseudovirions (f). g, h Transduction of 293 cells expressing human ACE2, R. malayanus ACE2, or P. abramus ACE2 by WT and Q493K mutant BANAL-20-52 S pseudovirions (g), and WT and K489Q mutant BANAL-20-236 S pseudovirions (h). Data are represented as means ± SD from at least triplicates. P-values in e–h are calculated by unpaired two-sided Student’s t-test. *P < 0.05; **P < 0.01; ns, P > 0.05.