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. Author manuscript; available in PMC: 2023 Apr 25.
Published in final edited form as: Nat Chem Biol. 2022 Sep 29;19(3):275–283. doi: 10.1038/s41589-022-01149-6

Fig. 1 |. SNOC increases S-nitrosylation of ACE2 and inhibits binding of SARS-CoV-2 Spike (S) protein.

Fig. 1 |

a, Assay for SNO-ACE2 and SNO-TMPRSS2 in HeLa-ACE2 cells. Cells were exposed to 100 μM SNOC or, as a control, ‘old’ SNOC (from which NO had been dissipated). After 20 minutes, cell lysates were subjected to biotin-switch assay to assess S-nitrosylated (SNO-) and input (total) proteins detected by immunoblotting with cognate antibody. The ascorbate minus (Asc-) sample served as a negative control. b, c, Ratio of SNO-ACE2/input ACE2 protein and SNO-TMPRSS2/input TMPRSS2 protein. Data are mean + s.e.m.by one-way ANOVA with Tukey’s multiple comparisons. n = 3 biological replicates. d, HeLa and HeLa-ACE2 cells were pre-exposed to 100 μM SNOC or old SNOC. After 30 minutes, 10 μg/ml of purified recombinant SARS-CoV-2 Spike (S1 + S2) protein was incubated with the cells. After 1 h, cells were fixed with 4% PFA for 15 minutes, and bound Spike protein was detected by anti-Spike protein antibody; nuclei stained with Hoechst. Scale bar, 20 μm. e, Quantification of relative fluorescence intensity. Data are mean + s.e.m. by two-way ANOVA with Tukey’s multiple comparisons. n = 3 biological replicates.