USP50 Antibody - #AF9225

Figure 3. VitC‐induced reduction of ACE2 levels largely depends on the deubiquitinase USP50 A. Western blot analysis of ACE2 in HEK293T cells pretreated with PR619 (50 μM, 2 h) and then treated with VitC (5 mM) for 12 h. Data are representative of three biological replicates. B. HEK293T cells were individually transfected with the plasmids from the human DUBs expression library. Western blot was used to identify the key deubiquitinase that significantly increases ACE2 levels. Data were from two biological replicates and were shown as the fold change (the intensity of ACE2/Tubulin bands measured by the Image J) normalized to the empty vector group (CON). C. IP‐IB analysis of the interaction between Flag‐USP50 and Myc‐ACE2 in HEK293T cells cotransfected with these two constructs. Data are representative of three biological replicates. D. Immunoprecipitation analysis of the interaction between endogenous USP50 and ACE2 in 2fTGH cells. Data are representative of three biological replicates. E. Western blot analysis of ACE2 in HEK293T cells transfected with increasing amounts of Flag‐USP50. Data are representative of three biological replicates. F. Western blot analysis of ACE2 in HEK293T cells transfected with shCtrl or shUSP50 (#1 or #2). Data are representative of three biological replicates. G. Western blot analysis of ACE2 in HEK293T cells transfected with Flag‐USP50 and then treated with CHX (50 μM) as indicated. Data are representative of three biological replicates. H. Western blot analysis of ACE2 in Usp50+/+ and Usp50−/− HEK293T cells. Data are representative of three biological replicates. I. Western blot analysis of ACE2 in Usp50+/+ and Usp50−/− HEK293T cells treated with VitC (5 mM) for 12 h. Data are representative of three biological replicates. J. RT–qPCR analysis of SARS‐CoV‐2 GFP/ΔN RNA levels in HEK293T cells transfected with Flag‐USP50 and then infected with the SARS‐CoV‐2 GFP/ΔN virus (MOI = 0.1) for 2 h. Data are shown as mean and s.d. of three biological replicates (n = 3). *P