<p>Pseudotype virus titrations and protein expression of ACE2s and sarbecovirus Spikes.</p>

<p><b>(A)</b> Lentiviral pseudotypes expressing different bat sarbecovirus Spikes were titrated on BHK-21 cells overexpressing human ACE2 (SARS-CoV-2, BANAL-20–52, BANAL-20–103, BANAL-20–236, RaTG13, SARS-CoV-1, WIV-1, Rs4231, Rf1, Rp3, RacCS203, RpYN06), <i>Rh. cornutus</i> ACE2 (Rc-o319) or <i>Rh. alcyone</i> ACE2 (RhGB07, RfGB02). Virus titres were used to normalise input virus for subsequent assays. <b>(B)</b> Expression of tagged ACE2 proteins was carried out in lysates from BHK-21 cells transfected with ACE2 constructs and probed for HA expression by Western blotting. <b>(C)</b> Sarbecovirus Spikes were used to generate lentivral-based pseudotypes, and the viral supernatant purified by ultracentrifugation under a 20% sucrose cushion.

Purified viruses were probed with an anti-FLAG antibody (except RhGB07 and RfGB02, which are untagged), an anti-Spike antibody (S, full-length Spike; S1/S2, cleaved variant) and the lentivirus internal control, anti-p24 antibody. The data underlying this Figure can be found in <a href="plosbiology.org/article/info:doi/10.1371/journal.pbio.3003944#pbio.3003944.s017" target="_blank">S1 Data</a> and <a href="plosbiology.org/article/info:doi/10.1371/journal.pbio.3003944#pbio.3003944.s018" target="_blank">S1 Raw Images</a>.</p> <p>(TIF)</p>