anti human ace2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti human ace2
    (A) Western blot analysis of endogenous <t>ACE2</t> in various types of cell lines, including HEK293T transfected with either control shRNAs (shCtrl) or shRNAs against ACE2 (shACE2), 2fTGH, Caco-2 and A549 cells. (B) HEK293T cells were transfected with shCtrl or increasing amounts of shACE2. Then cells were subjected to RT-qPCR analysis of Ace2 mRNA levels (right), or were infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) for 2 hrs. RT-qPCR was used to analyze SARS-CoV-2 RNA levels (left). (C) Western blot analysis of ACE2 in Caco-2 cells treated with vitamin (Vit) compounds (VitB1, 500 µM; VitB6, 500 µM; VitB12, 50 nM; VitC, 5 mM; VitD3, 25 µM; VitK1, 0.5 µM) for 24 hrs. (D) Western blot analysis of ACE2 in HEK293T, 2fTGH, Caco-2 and A549 cells treated with VitC at indicated concentrations for 24 hrs. (E) Western blot analysis of ACE2 in HEK293T cells treated with 5 mM or 0.2 mM of VitC for different durations. (F) Immunofluorescence analysis of ACE2 proteins in HeLa cells treated with VitC (5 mM) for 24 hrs. DAPI was used for the nucleus. Scale bars, 1 μm. (G) Western blot analysis of ACE2, IRF3 and STAT1 in A549 cells treated with VitC at indicated concentrations for 24 hrs. (H) Fluorescence microscopy of the SARS-CoV-2 GFP/ΔN or VSV-GFP viruses in Caco-2-N cells pretreated with VitC (5 mM and 10 mM) for 24 hrs, and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV-GFP (MOI = 0.1) viruses for 24 hrs. Scale bar: 100 µm. (I) RT-qPCR analysis of SARS-CoV-2 GFP/ΔN or VSV RNA levels in Caco-2 cells pretreated with VitC as (H), and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV (MOI = 0.1) for 2 hrs. Data are representative of three independent experiments (A, C-F), or are shown as mean and s.d. of three biological replicates (B, I). N.S, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S1.
    Anti Human Ace2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti human ace2/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti human ace2 - by Bioz Stars, 2023-01
    94/100 stars

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    1) Product Images from "Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models"

    Article Title: Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

    Journal: bioRxiv

    doi: 10.1101/2022.07.14.499651

    (A) Western blot analysis of endogenous ACE2 in various types of cell lines, including HEK293T transfected with either control shRNAs (shCtrl) or shRNAs against ACE2 (shACE2), 2fTGH, Caco-2 and A549 cells. (B) HEK293T cells were transfected with shCtrl or increasing amounts of shACE2. Then cells were subjected to RT-qPCR analysis of Ace2 mRNA levels (right), or were infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) for 2 hrs. RT-qPCR was used to analyze SARS-CoV-2 RNA levels (left). (C) Western blot analysis of ACE2 in Caco-2 cells treated with vitamin (Vit) compounds (VitB1, 500 µM; VitB6, 500 µM; VitB12, 50 nM; VitC, 5 mM; VitD3, 25 µM; VitK1, 0.5 µM) for 24 hrs. (D) Western blot analysis of ACE2 in HEK293T, 2fTGH, Caco-2 and A549 cells treated with VitC at indicated concentrations for 24 hrs. (E) Western blot analysis of ACE2 in HEK293T cells treated with 5 mM or 0.2 mM of VitC for different durations. (F) Immunofluorescence analysis of ACE2 proteins in HeLa cells treated with VitC (5 mM) for 24 hrs. DAPI was used for the nucleus. Scale bars, 1 μm. (G) Western blot analysis of ACE2, IRF3 and STAT1 in A549 cells treated with VitC at indicated concentrations for 24 hrs. (H) Fluorescence microscopy of the SARS-CoV-2 GFP/ΔN or VSV-GFP viruses in Caco-2-N cells pretreated with VitC (5 mM and 10 mM) for 24 hrs, and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV-GFP (MOI = 0.1) viruses for 24 hrs. Scale bar: 100 µm. (I) RT-qPCR analysis of SARS-CoV-2 GFP/ΔN or VSV RNA levels in Caco-2 cells pretreated with VitC as (H), and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV (MOI = 0.1) for 2 hrs. Data are representative of three independent experiments (A, C-F), or are shown as mean and s.d. of three biological replicates (B, I). N.S, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S1.
    Figure Legend Snippet: (A) Western blot analysis of endogenous ACE2 in various types of cell lines, including HEK293T transfected with either control shRNAs (shCtrl) or shRNAs against ACE2 (shACE2), 2fTGH, Caco-2 and A549 cells. (B) HEK293T cells were transfected with shCtrl or increasing amounts of shACE2. Then cells were subjected to RT-qPCR analysis of Ace2 mRNA levels (right), or were infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) for 2 hrs. RT-qPCR was used to analyze SARS-CoV-2 RNA levels (left). (C) Western blot analysis of ACE2 in Caco-2 cells treated with vitamin (Vit) compounds (VitB1, 500 µM; VitB6, 500 µM; VitB12, 50 nM; VitC, 5 mM; VitD3, 25 µM; VitK1, 0.5 µM) for 24 hrs. (D) Western blot analysis of ACE2 in HEK293T, 2fTGH, Caco-2 and A549 cells treated with VitC at indicated concentrations for 24 hrs. (E) Western blot analysis of ACE2 in HEK293T cells treated with 5 mM or 0.2 mM of VitC for different durations. (F) Immunofluorescence analysis of ACE2 proteins in HeLa cells treated with VitC (5 mM) for 24 hrs. DAPI was used for the nucleus. Scale bars, 1 μm. (G) Western blot analysis of ACE2, IRF3 and STAT1 in A549 cells treated with VitC at indicated concentrations for 24 hrs. (H) Fluorescence microscopy of the SARS-CoV-2 GFP/ΔN or VSV-GFP viruses in Caco-2-N cells pretreated with VitC (5 mM and 10 mM) for 24 hrs, and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV-GFP (MOI = 0.1) viruses for 24 hrs. Scale bar: 100 µm. (I) RT-qPCR analysis of SARS-CoV-2 GFP/ΔN or VSV RNA levels in Caco-2 cells pretreated with VitC as (H), and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV (MOI = 0.1) for 2 hrs. Data are representative of three independent experiments (A, C-F), or are shown as mean and s.d. of three biological replicates (B, I). N.S, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S1.

    Techniques Used: Western Blot, Transfection, Quantitative RT-PCR, Infection, Immunofluorescence, Fluorescence, Microscopy, Two Tailed Test

    (A) RT-qPCR analysis of Ace2 mRNA in 2fTGH cells treated with VitC (5 mM) as indicated. (B) Western blot analysis of ACE2 in 2fTGH cells pretreated with ddH 2 O (Ctrl) or VitC (5 mM) for 12 hrs and then treated with CHX (50 μM) for 6 and 12 hrs. (C) Western blot analysis of Myc-ACE2 levels in HEK293T cells transfected with Myc-ACE2 and then treated with VitC at indicated concentrations for 12 hrs. (D) Western blot analysis of ACE2 in HEK293T cells pretreated with MG132 (10 µM) or MA (10 µM) for 2 hrs, followed by VitC treatment (5 mM) for 6 hrs. (E) Immunoprecipitation (IP)-immunoblotting (IB) analysis of ubiquitination (Ub) of endogenous ACE2 in 2fTGH cells treated with VitC at indicated concentrations for 12 hrs. (F) IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2 and different types of HA-Ub, and then treated with VitC (5 mM) for 12 hrs. (G) IP-IB analysis of K48-linked polyubiquitination (K48-Ub) of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 and then treated with VitC (2.5 mM and 5 mM) for 12 hrs, using a specific anti-K48-Ub antibody. (H) IP-IB analysis of K48-Ub of endogenous ACE2 in 2fTGH cells treated with VitC (2.5 mM and 5 mM) for 12 hrs. Data are representative of three independent experiments (B-H), or are shown as mean and s.d. of three biological replicates (A, B). N.S, not significant, ** p < 0.01 (two-tailed unpaired Student’s t -test). See also Figure S2.
    Figure Legend Snippet: (A) RT-qPCR analysis of Ace2 mRNA in 2fTGH cells treated with VitC (5 mM) as indicated. (B) Western blot analysis of ACE2 in 2fTGH cells pretreated with ddH 2 O (Ctrl) or VitC (5 mM) for 12 hrs and then treated with CHX (50 μM) for 6 and 12 hrs. (C) Western blot analysis of Myc-ACE2 levels in HEK293T cells transfected with Myc-ACE2 and then treated with VitC at indicated concentrations for 12 hrs. (D) Western blot analysis of ACE2 in HEK293T cells pretreated with MG132 (10 µM) or MA (10 µM) for 2 hrs, followed by VitC treatment (5 mM) for 6 hrs. (E) Immunoprecipitation (IP)-immunoblotting (IB) analysis of ubiquitination (Ub) of endogenous ACE2 in 2fTGH cells treated with VitC at indicated concentrations for 12 hrs. (F) IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2 and different types of HA-Ub, and then treated with VitC (5 mM) for 12 hrs. (G) IP-IB analysis of K48-linked polyubiquitination (K48-Ub) of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 and then treated with VitC (2.5 mM and 5 mM) for 12 hrs, using a specific anti-K48-Ub antibody. (H) IP-IB analysis of K48-Ub of endogenous ACE2 in 2fTGH cells treated with VitC (2.5 mM and 5 mM) for 12 hrs. Data are representative of three independent experiments (B-H), or are shown as mean and s.d. of three biological replicates (A, B). N.S, not significant, ** p < 0.01 (two-tailed unpaired Student’s t -test). See also Figure S2.

    Techniques Used: Quantitative RT-PCR, Western Blot, Transfection, Immunoprecipitation, Two Tailed Test

    (A) Western blot analysis of ACE2 in HEK293T cells pretreated with PR619 (50 µM, 2 hrs) and then treated with VitC (5 mM) for 12 hrs. (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. (C) IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells cotransfected with these two constructs. (D) Immunoprecipitation analysis of the interaction between endogenous USP50 and ACE2 in 2fTGH cells. (E) Western blot analysis of ACE2 in HEK293T cells transfected with increasing amounts of Flag-USP50. (F) Western blot analysis of ACE2 in HEK293T cells transfected with shCtrl or shUSP50 (#1 or #2). (G) Western blot analysis of ACE2 in HEK293T cells transfected with Flag-USP50 and then treated with CHX (50 μM) as indicated. (H) Western blot analysis of ACE2 in Usp50 +/+ and Usp50 -/- HEK293T cells. (I) Western blot analysis of ACE2 in Usp50 +/+ and Usp50 -/- HEK293T cells treated with VitC (5 mM) for 12 hrs. (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 24 hrs. (K) Fluorescence microscopy of the SARS-CoV-2-S pseudovirus in Usp50 +/+ and Usp50 -/- HEK293T cells pretreated with or without VitC (5 mM) for 12 hrs, followed by infection with SARS-CoV-2-S pseudovirus (MOI = 0.1) for 24 hrs. Scale bar: 100 µm. Data are representative of three independent experiments (A-I), or are shown as mean and s.d. of three biological replicates (J, K). N.S, not significant, * p < 0.05, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S3.
    Figure Legend Snippet: (A) Western blot analysis of ACE2 in HEK293T cells pretreated with PR619 (50 µM, 2 hrs) and then treated with VitC (5 mM) for 12 hrs. (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. (C) IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells cotransfected with these two constructs. (D) Immunoprecipitation analysis of the interaction between endogenous USP50 and ACE2 in 2fTGH cells. (E) Western blot analysis of ACE2 in HEK293T cells transfected with increasing amounts of Flag-USP50. (F) Western blot analysis of ACE2 in HEK293T cells transfected with shCtrl or shUSP50 (#1 or #2). (G) Western blot analysis of ACE2 in HEK293T cells transfected with Flag-USP50 and then treated with CHX (50 μM) as indicated. (H) Western blot analysis of ACE2 in Usp50 +/+ and Usp50 -/- HEK293T cells. (I) Western blot analysis of ACE2 in Usp50 +/+ and Usp50 -/- HEK293T cells treated with VitC (5 mM) for 12 hrs. (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 24 hrs. (K) Fluorescence microscopy of the SARS-CoV-2-S pseudovirus in Usp50 +/+ and Usp50 -/- HEK293T cells pretreated with or without VitC (5 mM) for 12 hrs, followed by infection with SARS-CoV-2-S pseudovirus (MOI = 0.1) for 24 hrs. Scale bar: 100 µm. Data are representative of three independent experiments (A-I), or are shown as mean and s.d. of three biological replicates (J, K). N.S, not significant, * p < 0.05, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S3.

    Techniques Used: Western Blot, Transfection, Expressing, Construct, Immunoprecipitation, Quantitative RT-PCR, Infection, Fluorescence, Microscopy, Two Tailed Test

    (A) IP-IB analysis of ubiquitination of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2, together with Flag-USP50 (WT) or its deubiquitinase inactive mutant (C53S). (B) IP-IB analysis of ubiquitination of endogenous ACE2 in HEK293T cells transfected with either shCtrl (-) or shUSP50 (#1, #2). (C) IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2, Flag-USP50 and different types of HA-Ub. (D) IP-IB analysis of K48-Ub of endogenous ACE2 in HEK293T cells transfected with shCtrl (-) or shUSP50 (#1, #2). (E) Putative ubiquitination sites of ACE2 in the PhosphoSitePlus database (Upper). Myc-ACE2 K48-linked ubiquitination was analyzed by IP-IB in HEK293T cells cotransfected with Myc-ACE2 (WT or its mutants) and HA-K48-Ub (Lower). (F) IP-IB analysis of Myc-ACE2 K48-linked ubiquitination in HEK293T cells cotransfected with Myc-ACE2 (WT or K788R) and HA-K48, together with shCtrl or shUSP50. (G) Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with CHX (50 μM) as indicated. (H) IP-IB analysis of K48-Ub of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) for 12 hrs, by a specific anti-K48-Ub antibody. (I) Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) as indicated. Data are representative of three independent experiments (A-I). See also Figure S3 and S4.
    Figure Legend Snippet: (A) IP-IB analysis of ubiquitination of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2, together with Flag-USP50 (WT) or its deubiquitinase inactive mutant (C53S). (B) IP-IB analysis of ubiquitination of endogenous ACE2 in HEK293T cells transfected with either shCtrl (-) or shUSP50 (#1, #2). (C) IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2, Flag-USP50 and different types of HA-Ub. (D) IP-IB analysis of K48-Ub of endogenous ACE2 in HEK293T cells transfected with shCtrl (-) or shUSP50 (#1, #2). (E) Putative ubiquitination sites of ACE2 in the PhosphoSitePlus database (Upper). Myc-ACE2 K48-linked ubiquitination was analyzed by IP-IB in HEK293T cells cotransfected with Myc-ACE2 (WT or its mutants) and HA-K48-Ub (Lower). (F) IP-IB analysis of Myc-ACE2 K48-linked ubiquitination in HEK293T cells cotransfected with Myc-ACE2 (WT or K788R) and HA-K48, together with shCtrl or shUSP50. (G) Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with CHX (50 μM) as indicated. (H) IP-IB analysis of K48-Ub of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) for 12 hrs, by a specific anti-K48-Ub antibody. (I) Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) as indicated. Data are representative of three independent experiments (A-I). See also Figure S3 and S4.

    Techniques Used: Transfection, Mutagenesis, Western Blot

    (A) The procedure for analysis of the binding of VitC to Flag-USP50 or Myc-ACE2 (left) can be seen detailedly in the Methods. The concentrations of VitC that binds with either Flag-USP50 (middle) or Myc-ACE2 (right) proteins were measured by the Vitamin-C Detection Kit. (B) Recombinant human ACE2-IgG-Fc proteins (r-hACE2-Fc) or anti-Tyk2 IgG proteins were incubated with protein-G beads for 2 hrs, and then VitC was added for binding. After washing and centrifuging, the concentrations of VitC that binds to anti-Tyk2 proteins or r-hACE2-Fc proteins were detected as (A). (C) The concentrations of VitC that binds to Myc-ACE2 (WT) or its deletion mutants were detected as (A). (D) VitC docking to the binding pocket of ACE2 by standard precision (SP) using the Glide docking module in the Schrödinger molecular simulation software. (E) The docking diagram between amino acids of ACE2 and VitC based on the SP docking scoring function. (F) IP-IB analysis of the interaction between USP50 and Myc-ACE2 (WT) or its deletion mutants (Δ19-200, Δ201-400, Δ401-600 and Δ601-805) in HEK293T. (G) IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells transfected with these two constructs and then treated with VitC (2.5 mM and 5 mM) for 12 hrs. (H) IP-IB analysis of the in vivo interaction between endogenous USP50 and ACE2 in 2fTGH cells treated with VitC (5 mM) for 12 hrs. (I) Myc-ACE2 and Flag-USP50 proteins were immunoprecipitated from HEK293T cells transfected with either Myc-ACE2 or Flag-USP50. Flag-USP50 proteins were eluted by the Flag (M2) agarose. After washing, Flag-USP50 proteins were mixed with the Myc beads with Myc-ACE2, together with or without VitC (5 mM) for 2 hrs. After centrifuging, Flag-USP50 proteins interacting with Myc-ACE2 were analyzed by immunoblotting. (J) Flag-USP50 proteins were obtained as (I). Flag-USP50 and r-hACE2 proteins were mixed, together with increasing amounts of VitC or with VitC-Na + (20 mM). HCl is a pH control (pH = 4). After 2 hrs incubation, ACE2 proteins were analyzed by immunoblotting by a specific anti-ACE2 antibody. Data are representative of three independent experiments (F-J), or are shown as mean and s.d. of three biological replicates (A-C). N.S, not significant. *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S5.
    Figure Legend Snippet: (A) The procedure for analysis of the binding of VitC to Flag-USP50 or Myc-ACE2 (left) can be seen detailedly in the Methods. The concentrations of VitC that binds with either Flag-USP50 (middle) or Myc-ACE2 (right) proteins were measured by the Vitamin-C Detection Kit. (B) Recombinant human ACE2-IgG-Fc proteins (r-hACE2-Fc) or anti-Tyk2 IgG proteins were incubated with protein-G beads for 2 hrs, and then VitC was added for binding. After washing and centrifuging, the concentrations of VitC that binds to anti-Tyk2 proteins or r-hACE2-Fc proteins were detected as (A). (C) The concentrations of VitC that binds to Myc-ACE2 (WT) or its deletion mutants were detected as (A). (D) VitC docking to the binding pocket of ACE2 by standard precision (SP) using the Glide docking module in the Schrödinger molecular simulation software. (E) The docking diagram between amino acids of ACE2 and VitC based on the SP docking scoring function. (F) IP-IB analysis of the interaction between USP50 and Myc-ACE2 (WT) or its deletion mutants (Δ19-200, Δ201-400, Δ401-600 and Δ601-805) in HEK293T. (G) IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells transfected with these two constructs and then treated with VitC (2.5 mM and 5 mM) for 12 hrs. (H) IP-IB analysis of the in vivo interaction between endogenous USP50 and ACE2 in 2fTGH cells treated with VitC (5 mM) for 12 hrs. (I) Myc-ACE2 and Flag-USP50 proteins were immunoprecipitated from HEK293T cells transfected with either Myc-ACE2 or Flag-USP50. Flag-USP50 proteins were eluted by the Flag (M2) agarose. After washing, Flag-USP50 proteins were mixed with the Myc beads with Myc-ACE2, together with or without VitC (5 mM) for 2 hrs. After centrifuging, Flag-USP50 proteins interacting with Myc-ACE2 were analyzed by immunoblotting. (J) Flag-USP50 proteins were obtained as (I). Flag-USP50 and r-hACE2 proteins were mixed, together with increasing amounts of VitC or with VitC-Na + (20 mM). HCl is a pH control (pH = 4). After 2 hrs incubation, ACE2 proteins were analyzed by immunoblotting by a specific anti-ACE2 antibody. Data are representative of three independent experiments (F-J), or are shown as mean and s.d. of three biological replicates (A-C). N.S, not significant. *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S5.

    Techniques Used: Binding Assay, Recombinant, Incubation, Software, Transfection, Construct, In Vivo, Immunoprecipitation, Western Blot, Two Tailed Test

    (A) IP-IB analysis of the interaction between endogenous hACE2 and USP50 in the lung and liver tissues of hACE2 mice. (B) The hACE2 mice were intraperitoneally administrated with VitC (300 mg/day/kg body weight) for two days. The interaction between USP50 and hACE2 in mouse lung tissues was analyzed by IP-IB. (C) IP-IB analysis of K48-Ub of hACE2 in mouse lung tissues from (B). (D) Western blot analysis of hACE2 levels in lung tissues of hACE2 mice administrated with VitC as (B). (E) Immunohistochemical staining of hACE2 protein in the lung, kidney and liver tissues from (B). (F) The hACE2 mice were administrated with VitC as (B). Mice were then given intraperitoneal injections of SARS-CoV-2-S pseudoviruses (1×10 6 PFU per gram body). After 24 hrs, immunohistochemical staining was performed to analyze the SARS-CoV-2 Spike proteins in mouse lung and kidney tissues. Scale bar: 100 µm. (G) RT-qPCR analysis of the SARS-CoV-2 Spike mRNA levels in lung, kidney, liver and spleen tissues of hACE2 mice treated with VitC and SARS-CoV-2-S pseudoviruses as (F). Data are representative of three independent experiments (A-D). All graphs show the mean ± SEM for five individual mice (G). *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S6 and S7.
    Figure Legend Snippet: (A) IP-IB analysis of the interaction between endogenous hACE2 and USP50 in the lung and liver tissues of hACE2 mice. (B) The hACE2 mice were intraperitoneally administrated with VitC (300 mg/day/kg body weight) for two days. The interaction between USP50 and hACE2 in mouse lung tissues was analyzed by IP-IB. (C) IP-IB analysis of K48-Ub of hACE2 in mouse lung tissues from (B). (D) Western blot analysis of hACE2 levels in lung tissues of hACE2 mice administrated with VitC as (B). (E) Immunohistochemical staining of hACE2 protein in the lung, kidney and liver tissues from (B). (F) The hACE2 mice were administrated with VitC as (B). Mice were then given intraperitoneal injections of SARS-CoV-2-S pseudoviruses (1×10 6 PFU per gram body). After 24 hrs, immunohistochemical staining was performed to analyze the SARS-CoV-2 Spike proteins in mouse lung and kidney tissues. Scale bar: 100 µm. (G) RT-qPCR analysis of the SARS-CoV-2 Spike mRNA levels in lung, kidney, liver and spleen tissues of hACE2 mice treated with VitC and SARS-CoV-2-S pseudoviruses as (F). Data are representative of three independent experiments (A-D). All graphs show the mean ± SEM for five individual mice (G). *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S6 and S7.

    Techniques Used: Western Blot, Immunohistochemical staining, Staining, Quantitative RT-PCR, Two Tailed Test

    anti human ace2  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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  • 94

    Structured Review

    Cell Signaling Technology Inc anti human ace2
    (A) Western blot analysis of endogenous <t>ACE2</t> in various types of cell lines, including HEK293T transfected with either control shRNAs (shCtrl) or shRNAs against ACE2 (shACE2), 2fTGH, Caco-2 and A549 cells. (B) HEK293T cells were transfected with shCtrl or increasing amounts of shACE2. Then cells were subjected to RT-qPCR analysis of Ace2 mRNA levels (right), or were infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) for 2 hrs. RT-qPCR was used to analyze SARS-CoV-2 RNA levels (left). (C) Western blot analysis of ACE2 in Caco-2 cells treated with vitamin (Vit) compounds (VitB1, 500 µM; VitB6, 500 µM; VitB12, 50 nM; VitC, 5 mM; VitD3, 25 µM; VitK1, 0.5 µM) for 24 hrs. (D) Western blot analysis of ACE2 in HEK293T, 2fTGH, Caco-2 and A549 cells treated with VitC at indicated concentrations for 24 hrs. (E) Western blot analysis of ACE2 in HEK293T cells treated with 5 mM or 0.2 mM of VitC for different durations. (F) Immunofluorescence analysis of ACE2 proteins in HeLa cells treated with VitC (5 mM) for 24 hrs. DAPI was used for the nucleus. Scale bars, 1 μm. (G) Western blot analysis of ACE2, IRF3 and STAT1 in A549 cells treated with VitC at indicated concentrations for 24 hrs. (H) Fluorescence microscopy of the SARS-CoV-2 GFP/ΔN or VSV-GFP viruses in Caco-2-N cells pretreated with VitC (5 mM and 10 mM) for 24 hrs, and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV-GFP (MOI = 0.1) viruses for 24 hrs. Scale bar: 100 µm. (I) RT-qPCR analysis of SARS-CoV-2 GFP/ΔN or VSV RNA levels in Caco-2 cells pretreated with VitC as (H), and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV (MOI = 0.1) for 2 hrs. Data are representative of three independent experiments (A, C-F), or are shown as mean and s.d. of three biological replicates (B, I). N.S, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S1.
    Anti Human Ace2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti human ace2/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti human ace2 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models"

    Article Title: Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

    Journal: bioRxiv

    doi: 10.1101/2022.07.14.499651

    (A) Western blot analysis of endogenous ACE2 in various types of cell lines, including HEK293T transfected with either control shRNAs (shCtrl) or shRNAs against ACE2 (shACE2), 2fTGH, Caco-2 and A549 cells. (B) HEK293T cells were transfected with shCtrl or increasing amounts of shACE2. Then cells were subjected to RT-qPCR analysis of Ace2 mRNA levels (right), or were infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) for 2 hrs. RT-qPCR was used to analyze SARS-CoV-2 RNA levels (left). (C) Western blot analysis of ACE2 in Caco-2 cells treated with vitamin (Vit) compounds (VitB1, 500 µM; VitB6, 500 µM; VitB12, 50 nM; VitC, 5 mM; VitD3, 25 µM; VitK1, 0.5 µM) for 24 hrs. (D) Western blot analysis of ACE2 in HEK293T, 2fTGH, Caco-2 and A549 cells treated with VitC at indicated concentrations for 24 hrs. (E) Western blot analysis of ACE2 in HEK293T cells treated with 5 mM or 0.2 mM of VitC for different durations. (F) Immunofluorescence analysis of ACE2 proteins in HeLa cells treated with VitC (5 mM) for 24 hrs. DAPI was used for the nucleus. Scale bars, 1 μm. (G) Western blot analysis of ACE2, IRF3 and STAT1 in A549 cells treated with VitC at indicated concentrations for 24 hrs. (H) Fluorescence microscopy of the SARS-CoV-2 GFP/ΔN or VSV-GFP viruses in Caco-2-N cells pretreated with VitC (5 mM and 10 mM) for 24 hrs, and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV-GFP (MOI = 0.1) viruses for 24 hrs. Scale bar: 100 µm. (I) RT-qPCR analysis of SARS-CoV-2 GFP/ΔN or VSV RNA levels in Caco-2 cells pretreated with VitC as (H), and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV (MOI = 0.1) for 2 hrs. Data are representative of three independent experiments (A, C-F), or are shown as mean and s.d. of three biological replicates (B, I). N.S, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S1.
    Figure Legend Snippet: (A) Western blot analysis of endogenous ACE2 in various types of cell lines, including HEK293T transfected with either control shRNAs (shCtrl) or shRNAs against ACE2 (shACE2), 2fTGH, Caco-2 and A549 cells. (B) HEK293T cells were transfected with shCtrl or increasing amounts of shACE2. Then cells were subjected to RT-qPCR analysis of Ace2 mRNA levels (right), or were infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) for 2 hrs. RT-qPCR was used to analyze SARS-CoV-2 RNA levels (left). (C) Western blot analysis of ACE2 in Caco-2 cells treated with vitamin (Vit) compounds (VitB1, 500 µM; VitB6, 500 µM; VitB12, 50 nM; VitC, 5 mM; VitD3, 25 µM; VitK1, 0.5 µM) for 24 hrs. (D) Western blot analysis of ACE2 in HEK293T, 2fTGH, Caco-2 and A549 cells treated with VitC at indicated concentrations for 24 hrs. (E) Western blot analysis of ACE2 in HEK293T cells treated with 5 mM or 0.2 mM of VitC for different durations. (F) Immunofluorescence analysis of ACE2 proteins in HeLa cells treated with VitC (5 mM) for 24 hrs. DAPI was used for the nucleus. Scale bars, 1 μm. (G) Western blot analysis of ACE2, IRF3 and STAT1 in A549 cells treated with VitC at indicated concentrations for 24 hrs. (H) Fluorescence microscopy of the SARS-CoV-2 GFP/ΔN or VSV-GFP viruses in Caco-2-N cells pretreated with VitC (5 mM and 10 mM) for 24 hrs, and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV-GFP (MOI = 0.1) viruses for 24 hrs. Scale bar: 100 µm. (I) RT-qPCR analysis of SARS-CoV-2 GFP/ΔN or VSV RNA levels in Caco-2 cells pretreated with VitC as (H), and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV (MOI = 0.1) for 2 hrs. Data are representative of three independent experiments (A, C-F), or are shown as mean and s.d. of three biological replicates (B, I). N.S, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S1.

    Techniques Used: Western Blot, Transfection, Quantitative RT-PCR, Infection, Immunofluorescence, Fluorescence, Microscopy, Two Tailed Test

    (A) RT-qPCR analysis of Ace2 mRNA in 2fTGH cells treated with VitC (5 mM) as indicated. (B) Western blot analysis of ACE2 in 2fTGH cells pretreated with ddH 2 O (Ctrl) or VitC (5 mM) for 12 hrs and then treated with CHX (50 μM) for 6 and 12 hrs. (C) Western blot analysis of Myc-ACE2 levels in HEK293T cells transfected with Myc-ACE2 and then treated with VitC at indicated concentrations for 12 hrs. (D) Western blot analysis of ACE2 in HEK293T cells pretreated with MG132 (10 µM) or MA (10 µM) for 2 hrs, followed by VitC treatment (5 mM) for 6 hrs. (E) Immunoprecipitation (IP)-immunoblotting (IB) analysis of ubiquitination (Ub) of endogenous ACE2 in 2fTGH cells treated with VitC at indicated concentrations for 12 hrs. (F) IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2 and different types of HA-Ub, and then treated with VitC (5 mM) for 12 hrs. (G) IP-IB analysis of K48-linked polyubiquitination (K48-Ub) of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 and then treated with VitC (2.5 mM and 5 mM) for 12 hrs, using a specific anti-K48-Ub antibody. (H) IP-IB analysis of K48-Ub of endogenous ACE2 in 2fTGH cells treated with VitC (2.5 mM and 5 mM) for 12 hrs. Data are representative of three independent experiments (B-H), or are shown as mean and s.d. of three biological replicates (A, B). N.S, not significant, ** p < 0.01 (two-tailed unpaired Student’s t -test). See also Figure S2.
    Figure Legend Snippet: (A) RT-qPCR analysis of Ace2 mRNA in 2fTGH cells treated with VitC (5 mM) as indicated. (B) Western blot analysis of ACE2 in 2fTGH cells pretreated with ddH 2 O (Ctrl) or VitC (5 mM) for 12 hrs and then treated with CHX (50 μM) for 6 and 12 hrs. (C) Western blot analysis of Myc-ACE2 levels in HEK293T cells transfected with Myc-ACE2 and then treated with VitC at indicated concentrations for 12 hrs. (D) Western blot analysis of ACE2 in HEK293T cells pretreated with MG132 (10 µM) or MA (10 µM) for 2 hrs, followed by VitC treatment (5 mM) for 6 hrs. (E) Immunoprecipitation (IP)-immunoblotting (IB) analysis of ubiquitination (Ub) of endogenous ACE2 in 2fTGH cells treated with VitC at indicated concentrations for 12 hrs. (F) IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2 and different types of HA-Ub, and then treated with VitC (5 mM) for 12 hrs. (G) IP-IB analysis of K48-linked polyubiquitination (K48-Ub) of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 and then treated with VitC (2.5 mM and 5 mM) for 12 hrs, using a specific anti-K48-Ub antibody. (H) IP-IB analysis of K48-Ub of endogenous ACE2 in 2fTGH cells treated with VitC (2.5 mM and 5 mM) for 12 hrs. Data are representative of three independent experiments (B-H), or are shown as mean and s.d. of three biological replicates (A, B). N.S, not significant, ** p < 0.01 (two-tailed unpaired Student’s t -test). See also Figure S2.

    Techniques Used: Quantitative RT-PCR, Western Blot, Transfection, Immunoprecipitation, Two Tailed Test

    (A) Western blot analysis of ACE2 in HEK293T cells pretreated with PR619 (50 µM, 2 hrs) and then treated with VitC (5 mM) for 12 hrs. (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. (C) IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells cotransfected with these two constructs. (D) Immunoprecipitation analysis of the interaction between endogenous USP50 and ACE2 in 2fTGH cells. (E) Western blot analysis of ACE2 in HEK293T cells transfected with increasing amounts of Flag-USP50. (F) Western blot analysis of ACE2 in HEK293T cells transfected with shCtrl or shUSP50 (#1 or #2). (G) Western blot analysis of ACE2 in HEK293T cells transfected with Flag-USP50 and then treated with CHX (50 μM) as indicated. (H) Western blot analysis of ACE2 in Usp50 +/+ and Usp50 -/- HEK293T cells. (I) Western blot analysis of ACE2 in Usp50 +/+ and Usp50 -/- HEK293T cells treated with VitC (5 mM) for 12 hrs. (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 24 hrs. (K) Fluorescence microscopy of the SARS-CoV-2-S pseudovirus in Usp50 +/+ and Usp50 -/- HEK293T cells pretreated with or without VitC (5 mM) for 12 hrs, followed by infection with SARS-CoV-2-S pseudovirus (MOI = 0.1) for 24 hrs. Scale bar: 100 µm. Data are representative of three independent experiments (A-I), or are shown as mean and s.d. of three biological replicates (J, K). N.S, not significant, * p < 0.05, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S3.
    Figure Legend Snippet: (A) Western blot analysis of ACE2 in HEK293T cells pretreated with PR619 (50 µM, 2 hrs) and then treated with VitC (5 mM) for 12 hrs. (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. (C) IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells cotransfected with these two constructs. (D) Immunoprecipitation analysis of the interaction between endogenous USP50 and ACE2 in 2fTGH cells. (E) Western blot analysis of ACE2 in HEK293T cells transfected with increasing amounts of Flag-USP50. (F) Western blot analysis of ACE2 in HEK293T cells transfected with shCtrl or shUSP50 (#1 or #2). (G) Western blot analysis of ACE2 in HEK293T cells transfected with Flag-USP50 and then treated with CHX (50 μM) as indicated. (H) Western blot analysis of ACE2 in Usp50 +/+ and Usp50 -/- HEK293T cells. (I) Western blot analysis of ACE2 in Usp50 +/+ and Usp50 -/- HEK293T cells treated with VitC (5 mM) for 12 hrs. (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 24 hrs. (K) Fluorescence microscopy of the SARS-CoV-2-S pseudovirus in Usp50 +/+ and Usp50 -/- HEK293T cells pretreated with or without VitC (5 mM) for 12 hrs, followed by infection with SARS-CoV-2-S pseudovirus (MOI = 0.1) for 24 hrs. Scale bar: 100 µm. Data are representative of three independent experiments (A-I), or are shown as mean and s.d. of three biological replicates (J, K). N.S, not significant, * p < 0.05, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S3.

    Techniques Used: Western Blot, Transfection, Expressing, Construct, Immunoprecipitation, Quantitative RT-PCR, Infection, Fluorescence, Microscopy, Two Tailed Test

    (A) IP-IB analysis of ubiquitination of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2, together with Flag-USP50 (WT) or its deubiquitinase inactive mutant (C53S). (B) IP-IB analysis of ubiquitination of endogenous ACE2 in HEK293T cells transfected with either shCtrl (-) or shUSP50 (#1, #2). (C) IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2, Flag-USP50 and different types of HA-Ub. (D) IP-IB analysis of K48-Ub of endogenous ACE2 in HEK293T cells transfected with shCtrl (-) or shUSP50 (#1, #2). (E) Putative ubiquitination sites of ACE2 in the PhosphoSitePlus database (Upper). Myc-ACE2 K48-linked ubiquitination was analyzed by IP-IB in HEK293T cells cotransfected with Myc-ACE2 (WT or its mutants) and HA-K48-Ub (Lower). (F) IP-IB analysis of Myc-ACE2 K48-linked ubiquitination in HEK293T cells cotransfected with Myc-ACE2 (WT or K788R) and HA-K48, together with shCtrl or shUSP50. (G) Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with CHX (50 μM) as indicated. (H) IP-IB analysis of K48-Ub of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) for 12 hrs, by a specific anti-K48-Ub antibody. (I) Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) as indicated. Data are representative of three independent experiments (A-I). See also Figure S3 and S4.
    Figure Legend Snippet: (A) IP-IB analysis of ubiquitination of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2, together with Flag-USP50 (WT) or its deubiquitinase inactive mutant (C53S). (B) IP-IB analysis of ubiquitination of endogenous ACE2 in HEK293T cells transfected with either shCtrl (-) or shUSP50 (#1, #2). (C) IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2, Flag-USP50 and different types of HA-Ub. (D) IP-IB analysis of K48-Ub of endogenous ACE2 in HEK293T cells transfected with shCtrl (-) or shUSP50 (#1, #2). (E) Putative ubiquitination sites of ACE2 in the PhosphoSitePlus database (Upper). Myc-ACE2 K48-linked ubiquitination was analyzed by IP-IB in HEK293T cells cotransfected with Myc-ACE2 (WT or its mutants) and HA-K48-Ub (Lower). (F) IP-IB analysis of Myc-ACE2 K48-linked ubiquitination in HEK293T cells cotransfected with Myc-ACE2 (WT or K788R) and HA-K48, together with shCtrl or shUSP50. (G) Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with CHX (50 μM) as indicated. (H) IP-IB analysis of K48-Ub of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) for 12 hrs, by a specific anti-K48-Ub antibody. (I) Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) as indicated. Data are representative of three independent experiments (A-I). See also Figure S3 and S4.

    Techniques Used: Transfection, Mutagenesis, Western Blot

    (A) The procedure for analysis of the binding of VitC to Flag-USP50 or Myc-ACE2 (left) can be seen detailedly in the Methods. The concentrations of VitC that binds with either Flag-USP50 (middle) or Myc-ACE2 (right) proteins were measured by the Vitamin-C Detection Kit. (B) Recombinant human ACE2-IgG-Fc proteins (r-hACE2-Fc) or anti-Tyk2 IgG proteins were incubated with protein-G beads for 2 hrs, and then VitC was added for binding. After washing and centrifuging, the concentrations of VitC that binds to anti-Tyk2 proteins or r-hACE2-Fc proteins were detected as (A). (C) The concentrations of VitC that binds to Myc-ACE2 (WT) or its deletion mutants were detected as (A). (D) VitC docking to the binding pocket of ACE2 by standard precision (SP) using the Glide docking module in the Schrödinger molecular simulation software. (E) The docking diagram between amino acids of ACE2 and VitC based on the SP docking scoring function. (F) IP-IB analysis of the interaction between USP50 and Myc-ACE2 (WT) or its deletion mutants (Δ19-200, Δ201-400, Δ401-600 and Δ601-805) in HEK293T. (G) IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells transfected with these two constructs and then treated with VitC (2.5 mM and 5 mM) for 12 hrs. (H) IP-IB analysis of the in vivo interaction between endogenous USP50 and ACE2 in 2fTGH cells treated with VitC (5 mM) for 12 hrs. (I) Myc-ACE2 and Flag-USP50 proteins were immunoprecipitated from HEK293T cells transfected with either Myc-ACE2 or Flag-USP50. Flag-USP50 proteins were eluted by the Flag (M2) agarose. After washing, Flag-USP50 proteins were mixed with the Myc beads with Myc-ACE2, together with or without VitC (5 mM) for 2 hrs. After centrifuging, Flag-USP50 proteins interacting with Myc-ACE2 were analyzed by immunoblotting. (J) Flag-USP50 proteins were obtained as (I). Flag-USP50 and r-hACE2 proteins were mixed, together with increasing amounts of VitC or with VitC-Na + (20 mM). HCl is a pH control (pH = 4). After 2 hrs incubation, ACE2 proteins were analyzed by immunoblotting by a specific anti-ACE2 antibody. Data are representative of three independent experiments (F-J), or are shown as mean and s.d. of three biological replicates (A-C). N.S, not significant. *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S5.
    Figure Legend Snippet: (A) The procedure for analysis of the binding of VitC to Flag-USP50 or Myc-ACE2 (left) can be seen detailedly in the Methods. The concentrations of VitC that binds with either Flag-USP50 (middle) or Myc-ACE2 (right) proteins were measured by the Vitamin-C Detection Kit. (B) Recombinant human ACE2-IgG-Fc proteins (r-hACE2-Fc) or anti-Tyk2 IgG proteins were incubated with protein-G beads for 2 hrs, and then VitC was added for binding. After washing and centrifuging, the concentrations of VitC that binds to anti-Tyk2 proteins or r-hACE2-Fc proteins were detected as (A). (C) The concentrations of VitC that binds to Myc-ACE2 (WT) or its deletion mutants were detected as (A). (D) VitC docking to the binding pocket of ACE2 by standard precision (SP) using the Glide docking module in the Schrödinger molecular simulation software. (E) The docking diagram between amino acids of ACE2 and VitC based on the SP docking scoring function. (F) IP-IB analysis of the interaction between USP50 and Myc-ACE2 (WT) or its deletion mutants (Δ19-200, Δ201-400, Δ401-600 and Δ601-805) in HEK293T. (G) IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells transfected with these two constructs and then treated with VitC (2.5 mM and 5 mM) for 12 hrs. (H) IP-IB analysis of the in vivo interaction between endogenous USP50 and ACE2 in 2fTGH cells treated with VitC (5 mM) for 12 hrs. (I) Myc-ACE2 and Flag-USP50 proteins were immunoprecipitated from HEK293T cells transfected with either Myc-ACE2 or Flag-USP50. Flag-USP50 proteins were eluted by the Flag (M2) agarose. After washing, Flag-USP50 proteins were mixed with the Myc beads with Myc-ACE2, together with or without VitC (5 mM) for 2 hrs. After centrifuging, Flag-USP50 proteins interacting with Myc-ACE2 were analyzed by immunoblotting. (J) Flag-USP50 proteins were obtained as (I). Flag-USP50 and r-hACE2 proteins were mixed, together with increasing amounts of VitC or with VitC-Na + (20 mM). HCl is a pH control (pH = 4). After 2 hrs incubation, ACE2 proteins were analyzed by immunoblotting by a specific anti-ACE2 antibody. Data are representative of three independent experiments (F-J), or are shown as mean and s.d. of three biological replicates (A-C). N.S, not significant. *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S5.

    Techniques Used: Binding Assay, Recombinant, Incubation, Software, Transfection, Construct, In Vivo, Immunoprecipitation, Western Blot, Two Tailed Test

    (A) IP-IB analysis of the interaction between endogenous hACE2 and USP50 in the lung and liver tissues of hACE2 mice. (B) The hACE2 mice were intraperitoneally administrated with VitC (300 mg/day/kg body weight) for two days. The interaction between USP50 and hACE2 in mouse lung tissues was analyzed by IP-IB. (C) IP-IB analysis of K48-Ub of hACE2 in mouse lung tissues from (B). (D) Western blot analysis of hACE2 levels in lung tissues of hACE2 mice administrated with VitC as (B). (E) Immunohistochemical staining of hACE2 protein in the lung, kidney and liver tissues from (B). (F) The hACE2 mice were administrated with VitC as (B). Mice were then given intraperitoneal injections of SARS-CoV-2-S pseudoviruses (1×10 6 PFU per gram body). After 24 hrs, immunohistochemical staining was performed to analyze the SARS-CoV-2 Spike proteins in mouse lung and kidney tissues. Scale bar: 100 µm. (G) RT-qPCR analysis of the SARS-CoV-2 Spike mRNA levels in lung, kidney, liver and spleen tissues of hACE2 mice treated with VitC and SARS-CoV-2-S pseudoviruses as (F). Data are representative of three independent experiments (A-D). All graphs show the mean ± SEM for five individual mice (G). *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S6 and S7.
    Figure Legend Snippet: (A) IP-IB analysis of the interaction between endogenous hACE2 and USP50 in the lung and liver tissues of hACE2 mice. (B) The hACE2 mice were intraperitoneally administrated with VitC (300 mg/day/kg body weight) for two days. The interaction between USP50 and hACE2 in mouse lung tissues was analyzed by IP-IB. (C) IP-IB analysis of K48-Ub of hACE2 in mouse lung tissues from (B). (D) Western blot analysis of hACE2 levels in lung tissues of hACE2 mice administrated with VitC as (B). (E) Immunohistochemical staining of hACE2 protein in the lung, kidney and liver tissues from (B). (F) The hACE2 mice were administrated with VitC as (B). Mice were then given intraperitoneal injections of SARS-CoV-2-S pseudoviruses (1×10 6 PFU per gram body). After 24 hrs, immunohistochemical staining was performed to analyze the SARS-CoV-2 Spike proteins in mouse lung and kidney tissues. Scale bar: 100 µm. (G) RT-qPCR analysis of the SARS-CoV-2 Spike mRNA levels in lung, kidney, liver and spleen tissues of hACE2 mice treated with VitC and SARS-CoV-2-S pseudoviruses as (F). Data are representative of three independent experiments (A-D). All graphs show the mean ± SEM for five individual mice (G). *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S6 and S7.

    Techniques Used: Western Blot, Immunohistochemical staining, Staining, Quantitative RT-PCR, Two Tailed Test

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    (A) Western blot analysis of endogenous <t>ACE2</t> in various types of cell lines, including HEK293T transfected with either control shRNAs (shCtrl) or shRNAs against ACE2 (shACE2), 2fTGH, Caco-2 and A549 cells. (B) HEK293T cells were transfected with shCtrl or increasing amounts of shACE2. Then cells were subjected to RT-qPCR analysis of Ace2 mRNA levels (right), or were infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) for 2 hrs. RT-qPCR was used to analyze SARS-CoV-2 RNA levels (left). (C) Western blot analysis of ACE2 in Caco-2 cells treated with vitamin (Vit) compounds (VitB1, 500 µM; VitB6, 500 µM; VitB12, 50 nM; VitC, 5 mM; VitD3, 25 µM; VitK1, 0.5 µM) for 24 hrs. (D) Western blot analysis of ACE2 in HEK293T, 2fTGH, Caco-2 and A549 cells treated with VitC at indicated concentrations for 24 hrs. (E) Western blot analysis of ACE2 in HEK293T cells treated with 5 mM or 0.2 mM of VitC for different durations. (F) Immunofluorescence analysis of ACE2 proteins in HeLa cells treated with VitC (5 mM) for 24 hrs. DAPI was used for the nucleus. Scale bars, 1 μm. (G) Western blot analysis of ACE2, IRF3 and STAT1 in A549 cells treated with VitC at indicated concentrations for 24 hrs. (H) Fluorescence microscopy of the SARS-CoV-2 GFP/ΔN or VSV-GFP viruses in Caco-2-N cells pretreated with VitC (5 mM and 10 mM) for 24 hrs, and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV-GFP (MOI = 0.1) viruses for 24 hrs. Scale bar: 100 µm. (I) RT-qPCR analysis of SARS-CoV-2 GFP/ΔN or VSV RNA levels in Caco-2 cells pretreated with VitC as (H), and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV (MOI = 0.1) for 2 hrs. Data are representative of three independent experiments (A, C-F), or are shown as mean and s.d. of three biological replicates (B, I). N.S, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S1.
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    1) Product Images from "Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models"

    Article Title: Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

    Journal: bioRxiv

    doi: 10.1101/2022.07.14.499651

    (A) Western blot analysis of endogenous ACE2 in various types of cell lines, including HEK293T transfected with either control shRNAs (shCtrl) or shRNAs against ACE2 (shACE2), 2fTGH, Caco-2 and A549 cells. (B) HEK293T cells were transfected with shCtrl or increasing amounts of shACE2. Then cells were subjected to RT-qPCR analysis of Ace2 mRNA levels (right), or were infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) for 2 hrs. RT-qPCR was used to analyze SARS-CoV-2 RNA levels (left). (C) Western blot analysis of ACE2 in Caco-2 cells treated with vitamin (Vit) compounds (VitB1, 500 µM; VitB6, 500 µM; VitB12, 50 nM; VitC, 5 mM; VitD3, 25 µM; VitK1, 0.5 µM) for 24 hrs. (D) Western blot analysis of ACE2 in HEK293T, 2fTGH, Caco-2 and A549 cells treated with VitC at indicated concentrations for 24 hrs. (E) Western blot analysis of ACE2 in HEK293T cells treated with 5 mM or 0.2 mM of VitC for different durations. (F) Immunofluorescence analysis of ACE2 proteins in HeLa cells treated with VitC (5 mM) for 24 hrs. DAPI was used for the nucleus. Scale bars, 1 μm. (G) Western blot analysis of ACE2, IRF3 and STAT1 in A549 cells treated with VitC at indicated concentrations for 24 hrs. (H) Fluorescence microscopy of the SARS-CoV-2 GFP/ΔN or VSV-GFP viruses in Caco-2-N cells pretreated with VitC (5 mM and 10 mM) for 24 hrs, and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV-GFP (MOI = 0.1) viruses for 24 hrs. Scale bar: 100 µm. (I) RT-qPCR analysis of SARS-CoV-2 GFP/ΔN or VSV RNA levels in Caco-2 cells pretreated with VitC as (H), and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV (MOI = 0.1) for 2 hrs. Data are representative of three independent experiments (A, C-F), or are shown as mean and s.d. of three biological replicates (B, I). N.S, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S1.
    Figure Legend Snippet: (A) Western blot analysis of endogenous ACE2 in various types of cell lines, including HEK293T transfected with either control shRNAs (shCtrl) or shRNAs against ACE2 (shACE2), 2fTGH, Caco-2 and A549 cells. (B) HEK293T cells were transfected with shCtrl or increasing amounts of shACE2. Then cells were subjected to RT-qPCR analysis of Ace2 mRNA levels (right), or were infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) for 2 hrs. RT-qPCR was used to analyze SARS-CoV-2 RNA levels (left). (C) Western blot analysis of ACE2 in Caco-2 cells treated with vitamin (Vit) compounds (VitB1, 500 µM; VitB6, 500 µM; VitB12, 50 nM; VitC, 5 mM; VitD3, 25 µM; VitK1, 0.5 µM) for 24 hrs. (D) Western blot analysis of ACE2 in HEK293T, 2fTGH, Caco-2 and A549 cells treated with VitC at indicated concentrations for 24 hrs. (E) Western blot analysis of ACE2 in HEK293T cells treated with 5 mM or 0.2 mM of VitC for different durations. (F) Immunofluorescence analysis of ACE2 proteins in HeLa cells treated with VitC (5 mM) for 24 hrs. DAPI was used for the nucleus. Scale bars, 1 μm. (G) Western blot analysis of ACE2, IRF3 and STAT1 in A549 cells treated with VitC at indicated concentrations for 24 hrs. (H) Fluorescence microscopy of the SARS-CoV-2 GFP/ΔN or VSV-GFP viruses in Caco-2-N cells pretreated with VitC (5 mM and 10 mM) for 24 hrs, and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV-GFP (MOI = 0.1) viruses for 24 hrs. Scale bar: 100 µm. (I) RT-qPCR analysis of SARS-CoV-2 GFP/ΔN or VSV RNA levels in Caco-2 cells pretreated with VitC as (H), and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV (MOI = 0.1) for 2 hrs. Data are representative of three independent experiments (A, C-F), or are shown as mean and s.d. of three biological replicates (B, I). N.S, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S1.

    Techniques Used: Western Blot, Transfection, Quantitative RT-PCR, Infection, Immunofluorescence, Fluorescence, Microscopy, Two Tailed Test

    (A) RT-qPCR analysis of Ace2 mRNA in 2fTGH cells treated with VitC (5 mM) as indicated. (B) Western blot analysis of ACE2 in 2fTGH cells pretreated with ddH 2 O (Ctrl) or VitC (5 mM) for 12 hrs and then treated with CHX (50 μM) for 6 and 12 hrs. (C) Western blot analysis of Myc-ACE2 levels in HEK293T cells transfected with Myc-ACE2 and then treated with VitC at indicated concentrations for 12 hrs. (D) Western blot analysis of ACE2 in HEK293T cells pretreated with MG132 (10 µM) or MA (10 µM) for 2 hrs, followed by VitC treatment (5 mM) for 6 hrs. (E) Immunoprecipitation (IP)-immunoblotting (IB) analysis of ubiquitination (Ub) of endogenous ACE2 in 2fTGH cells treated with VitC at indicated concentrations for 12 hrs. (F) IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2 and different types of HA-Ub, and then treated with VitC (5 mM) for 12 hrs. (G) IP-IB analysis of K48-linked polyubiquitination (K48-Ub) of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 and then treated with VitC (2.5 mM and 5 mM) for 12 hrs, using a specific anti-K48-Ub antibody. (H) IP-IB analysis of K48-Ub of endogenous ACE2 in 2fTGH cells treated with VitC (2.5 mM and 5 mM) for 12 hrs. Data are representative of three independent experiments (B-H), or are shown as mean and s.d. of three biological replicates (A, B). N.S, not significant, ** p < 0.01 (two-tailed unpaired Student’s t -test). See also Figure S2.
    Figure Legend Snippet: (A) RT-qPCR analysis of Ace2 mRNA in 2fTGH cells treated with VitC (5 mM) as indicated. (B) Western blot analysis of ACE2 in 2fTGH cells pretreated with ddH 2 O (Ctrl) or VitC (5 mM) for 12 hrs and then treated with CHX (50 μM) for 6 and 12 hrs. (C) Western blot analysis of Myc-ACE2 levels in HEK293T cells transfected with Myc-ACE2 and then treated with VitC at indicated concentrations for 12 hrs. (D) Western blot analysis of ACE2 in HEK293T cells pretreated with MG132 (10 µM) or MA (10 µM) for 2 hrs, followed by VitC treatment (5 mM) for 6 hrs. (E) Immunoprecipitation (IP)-immunoblotting (IB) analysis of ubiquitination (Ub) of endogenous ACE2 in 2fTGH cells treated with VitC at indicated concentrations for 12 hrs. (F) IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2 and different types of HA-Ub, and then treated with VitC (5 mM) for 12 hrs. (G) IP-IB analysis of K48-linked polyubiquitination (K48-Ub) of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 and then treated with VitC (2.5 mM and 5 mM) for 12 hrs, using a specific anti-K48-Ub antibody. (H) IP-IB analysis of K48-Ub of endogenous ACE2 in 2fTGH cells treated with VitC (2.5 mM and 5 mM) for 12 hrs. Data are representative of three independent experiments (B-H), or are shown as mean and s.d. of three biological replicates (A, B). N.S, not significant, ** p < 0.01 (two-tailed unpaired Student’s t -test). See also Figure S2.

    Techniques Used: Quantitative RT-PCR, Western Blot, Transfection, Immunoprecipitation, Two Tailed Test

    (A) Western blot analysis of ACE2 in HEK293T cells pretreated with PR619 (50 µM, 2 hrs) and then treated with VitC (5 mM) for 12 hrs. (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. (C) IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells cotransfected with these two constructs. (D) Immunoprecipitation analysis of the interaction between endogenous USP50 and ACE2 in 2fTGH cells. (E) Western blot analysis of ACE2 in HEK293T cells transfected with increasing amounts of Flag-USP50. (F) Western blot analysis of ACE2 in HEK293T cells transfected with shCtrl or shUSP50 (#1 or #2). (G) Western blot analysis of ACE2 in HEK293T cells transfected with Flag-USP50 and then treated with CHX (50 μM) as indicated. (H) Western blot analysis of ACE2 in Usp50 +/+ and Usp50 -/- HEK293T cells. (I) Western blot analysis of ACE2 in Usp50 +/+ and Usp50 -/- HEK293T cells treated with VitC (5 mM) for 12 hrs. (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 24 hrs. (K) Fluorescence microscopy of the SARS-CoV-2-S pseudovirus in Usp50 +/+ and Usp50 -/- HEK293T cells pretreated with or without VitC (5 mM) for 12 hrs, followed by infection with SARS-CoV-2-S pseudovirus (MOI = 0.1) for 24 hrs. Scale bar: 100 µm. Data are representative of three independent experiments (A-I), or are shown as mean and s.d. of three biological replicates (J, K). N.S, not significant, * p < 0.05, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S3.
    Figure Legend Snippet: (A) Western blot analysis of ACE2 in HEK293T cells pretreated with PR619 (50 µM, 2 hrs) and then treated with VitC (5 mM) for 12 hrs. (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. (C) IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells cotransfected with these two constructs. (D) Immunoprecipitation analysis of the interaction between endogenous USP50 and ACE2 in 2fTGH cells. (E) Western blot analysis of ACE2 in HEK293T cells transfected with increasing amounts of Flag-USP50. (F) Western blot analysis of ACE2 in HEK293T cells transfected with shCtrl or shUSP50 (#1 or #2). (G) Western blot analysis of ACE2 in HEK293T cells transfected with Flag-USP50 and then treated with CHX (50 μM) as indicated. (H) Western blot analysis of ACE2 in Usp50 +/+ and Usp50 -/- HEK293T cells. (I) Western blot analysis of ACE2 in Usp50 +/+ and Usp50 -/- HEK293T cells treated with VitC (5 mM) for 12 hrs. (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 24 hrs. (K) Fluorescence microscopy of the SARS-CoV-2-S pseudovirus in Usp50 +/+ and Usp50 -/- HEK293T cells pretreated with or without VitC (5 mM) for 12 hrs, followed by infection with SARS-CoV-2-S pseudovirus (MOI = 0.1) for 24 hrs. Scale bar: 100 µm. Data are representative of three independent experiments (A-I), or are shown as mean and s.d. of three biological replicates (J, K). N.S, not significant, * p < 0.05, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S3.

    Techniques Used: Western Blot, Transfection, Expressing, Construct, Immunoprecipitation, Quantitative RT-PCR, Infection, Fluorescence, Microscopy, Two Tailed Test

    (A) IP-IB analysis of ubiquitination of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2, together with Flag-USP50 (WT) or its deubiquitinase inactive mutant (C53S). (B) IP-IB analysis of ubiquitination of endogenous ACE2 in HEK293T cells transfected with either shCtrl (-) or shUSP50 (#1, #2). (C) IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2, Flag-USP50 and different types of HA-Ub. (D) IP-IB analysis of K48-Ub of endogenous ACE2 in HEK293T cells transfected with shCtrl (-) or shUSP50 (#1, #2). (E) Putative ubiquitination sites of ACE2 in the PhosphoSitePlus database (Upper). Myc-ACE2 K48-linked ubiquitination was analyzed by IP-IB in HEK293T cells cotransfected with Myc-ACE2 (WT or its mutants) and HA-K48-Ub (Lower). (F) IP-IB analysis of Myc-ACE2 K48-linked ubiquitination in HEK293T cells cotransfected with Myc-ACE2 (WT or K788R) and HA-K48, together with shCtrl or shUSP50. (G) Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with CHX (50 μM) as indicated. (H) IP-IB analysis of K48-Ub of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) for 12 hrs, by a specific anti-K48-Ub antibody. (I) Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) as indicated. Data are representative of three independent experiments (A-I). See also Figure S3 and S4.
    Figure Legend Snippet: (A) IP-IB analysis of ubiquitination of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2, together with Flag-USP50 (WT) or its deubiquitinase inactive mutant (C53S). (B) IP-IB analysis of ubiquitination of endogenous ACE2 in HEK293T cells transfected with either shCtrl (-) or shUSP50 (#1, #2). (C) IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2, Flag-USP50 and different types of HA-Ub. (D) IP-IB analysis of K48-Ub of endogenous ACE2 in HEK293T cells transfected with shCtrl (-) or shUSP50 (#1, #2). (E) Putative ubiquitination sites of ACE2 in the PhosphoSitePlus database (Upper). Myc-ACE2 K48-linked ubiquitination was analyzed by IP-IB in HEK293T cells cotransfected with Myc-ACE2 (WT or its mutants) and HA-K48-Ub (Lower). (F) IP-IB analysis of Myc-ACE2 K48-linked ubiquitination in HEK293T cells cotransfected with Myc-ACE2 (WT or K788R) and HA-K48, together with shCtrl or shUSP50. (G) Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with CHX (50 μM) as indicated. (H) IP-IB analysis of K48-Ub of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) for 12 hrs, by a specific anti-K48-Ub antibody. (I) Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) as indicated. Data are representative of three independent experiments (A-I). See also Figure S3 and S4.

    Techniques Used: Transfection, Mutagenesis, Western Blot

    (A) The procedure for analysis of the binding of VitC to Flag-USP50 or Myc-ACE2 (left) can be seen detailedly in the Methods. The concentrations of VitC that binds with either Flag-USP50 (middle) or Myc-ACE2 (right) proteins were measured by the Vitamin-C Detection Kit. (B) Recombinant human ACE2-IgG-Fc proteins (r-hACE2-Fc) or anti-Tyk2 IgG proteins were incubated with protein-G beads for 2 hrs, and then VitC was added for binding. After washing and centrifuging, the concentrations of VitC that binds to anti-Tyk2 proteins or r-hACE2-Fc proteins were detected as (A). (C) The concentrations of VitC that binds to Myc-ACE2 (WT) or its deletion mutants were detected as (A). (D) VitC docking to the binding pocket of ACE2 by standard precision (SP) using the Glide docking module in the Schrödinger molecular simulation software. (E) The docking diagram between amino acids of ACE2 and VitC based on the SP docking scoring function. (F) IP-IB analysis of the interaction between USP50 and Myc-ACE2 (WT) or its deletion mutants (Δ19-200, Δ201-400, Δ401-600 and Δ601-805) in HEK293T. (G) IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells transfected with these two constructs and then treated with VitC (2.5 mM and 5 mM) for 12 hrs. (H) IP-IB analysis of the in vivo interaction between endogenous USP50 and ACE2 in 2fTGH cells treated with VitC (5 mM) for 12 hrs. (I) Myc-ACE2 and Flag-USP50 proteins were immunoprecipitated from HEK293T cells transfected with either Myc-ACE2 or Flag-USP50. Flag-USP50 proteins were eluted by the Flag (M2) agarose. After washing, Flag-USP50 proteins were mixed with the Myc beads with Myc-ACE2, together with or without VitC (5 mM) for 2 hrs. After centrifuging, Flag-USP50 proteins interacting with Myc-ACE2 were analyzed by immunoblotting. (J) Flag-USP50 proteins were obtained as (I). Flag-USP50 and r-hACE2 proteins were mixed, together with increasing amounts of VitC or with VitC-Na + (20 mM). HCl is a pH control (pH = 4). After 2 hrs incubation, ACE2 proteins were analyzed by immunoblotting by a specific anti-ACE2 antibody. Data are representative of three independent experiments (F-J), or are shown as mean and s.d. of three biological replicates (A-C). N.S, not significant. *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S5.
    Figure Legend Snippet: (A) The procedure for analysis of the binding of VitC to Flag-USP50 or Myc-ACE2 (left) can be seen detailedly in the Methods. The concentrations of VitC that binds with either Flag-USP50 (middle) or Myc-ACE2 (right) proteins were measured by the Vitamin-C Detection Kit. (B) Recombinant human ACE2-IgG-Fc proteins (r-hACE2-Fc) or anti-Tyk2 IgG proteins were incubated with protein-G beads for 2 hrs, and then VitC was added for binding. After washing and centrifuging, the concentrations of VitC that binds to anti-Tyk2 proteins or r-hACE2-Fc proteins were detected as (A). (C) The concentrations of VitC that binds to Myc-ACE2 (WT) or its deletion mutants were detected as (A). (D) VitC docking to the binding pocket of ACE2 by standard precision (SP) using the Glide docking module in the Schrödinger molecular simulation software. (E) The docking diagram between amino acids of ACE2 and VitC based on the SP docking scoring function. (F) IP-IB analysis of the interaction between USP50 and Myc-ACE2 (WT) or its deletion mutants (Δ19-200, Δ201-400, Δ401-600 and Δ601-805) in HEK293T. (G) IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells transfected with these two constructs and then treated with VitC (2.5 mM and 5 mM) for 12 hrs. (H) IP-IB analysis of the in vivo interaction between endogenous USP50 and ACE2 in 2fTGH cells treated with VitC (5 mM) for 12 hrs. (I) Myc-ACE2 and Flag-USP50 proteins were immunoprecipitated from HEK293T cells transfected with either Myc-ACE2 or Flag-USP50. Flag-USP50 proteins were eluted by the Flag (M2) agarose. After washing, Flag-USP50 proteins were mixed with the Myc beads with Myc-ACE2, together with or without VitC (5 mM) for 2 hrs. After centrifuging, Flag-USP50 proteins interacting with Myc-ACE2 were analyzed by immunoblotting. (J) Flag-USP50 proteins were obtained as (I). Flag-USP50 and r-hACE2 proteins were mixed, together with increasing amounts of VitC or with VitC-Na + (20 mM). HCl is a pH control (pH = 4). After 2 hrs incubation, ACE2 proteins were analyzed by immunoblotting by a specific anti-ACE2 antibody. Data are representative of three independent experiments (F-J), or are shown as mean and s.d. of three biological replicates (A-C). N.S, not significant. *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S5.

    Techniques Used: Binding Assay, Recombinant, Incubation, Software, Transfection, Construct, In Vivo, Immunoprecipitation, Western Blot, Two Tailed Test

    ace2 oti1g4  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ace2 oti1g4
    Expression of SARS-CoV-2 entry factors in BRON and ALV organoids. (A) <t>ACE2</t> (left) and TMPRSS2 (right) expression is detected in patient L2 organoids of bronchiolar (BRON) and alveolar (ALV) differentiation from passage 1-4. Relative quantification was performed using the Livac method and alveolar organoids are normalized to the average delta Ct of bronchiolar organoids. The mean value of three technical replicates for each passage are shown as individual data points. GraphPadPrism v9 was used to calculate and display the geometric mean and 90% confidence interval of the biological replicates (passages). Statistics were performed on the delta Ct values using the Mann-Whitney test. No statistically significant differences were observed in the median deltaCt values of ACE2 between BRON and ALV cultures. (B) Immunofluorescent staining of formalin fixed paraffin embedded (FFPE) sections of bronchiolar (BRON) organoids (representative images from patient L2 passage 2), display co-staining of the cilia marker (ARL13B) and the SARS-CoV-2 receptor ACE2 (transparent arrowheads). ACE2 staining alone (white arrowheads). Scale bar equals 100 µm. (C) Immunofluorescent staining of formalin fixed paraffin embedded (FFPE) sections of alveolar (ALV) organoids (representative images from patient L2 passage 2), display co-staining of the cilia marker (ARL13B) and the SARS-CoV-2 receptor ACE2 (transparent arrowheads). Scale bar equals 100 µm. ns, not significant.
    Ace2 Oti1g4, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Human Organotypic Airway and Lung Organoid Cells of Bronchiolar and Alveolar Differentiation Are Permissive to Infection by Influenza and SARS-CoV-2 Respiratory Virus"

    Article Title: Human Organotypic Airway and Lung Organoid Cells of Bronchiolar and Alveolar Differentiation Are Permissive to Infection by Influenza and SARS-CoV-2 Respiratory Virus

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2022.841447

    Expression of SARS-CoV-2 entry factors in BRON and ALV organoids. (A) ACE2 (left) and TMPRSS2 (right) expression is detected in patient L2 organoids of bronchiolar (BRON) and alveolar (ALV) differentiation from passage 1-4. Relative quantification was performed using the Livac method and alveolar organoids are normalized to the average delta Ct of bronchiolar organoids. The mean value of three technical replicates for each passage are shown as individual data points. GraphPadPrism v9 was used to calculate and display the geometric mean and 90% confidence interval of the biological replicates (passages). Statistics were performed on the delta Ct values using the Mann-Whitney test. No statistically significant differences were observed in the median deltaCt values of ACE2 between BRON and ALV cultures. (B) Immunofluorescent staining of formalin fixed paraffin embedded (FFPE) sections of bronchiolar (BRON) organoids (representative images from patient L2 passage 2), display co-staining of the cilia marker (ARL13B) and the SARS-CoV-2 receptor ACE2 (transparent arrowheads). ACE2 staining alone (white arrowheads). Scale bar equals 100 µm. (C) Immunofluorescent staining of formalin fixed paraffin embedded (FFPE) sections of alveolar (ALV) organoids (representative images from patient L2 passage 2), display co-staining of the cilia marker (ARL13B) and the SARS-CoV-2 receptor ACE2 (transparent arrowheads). Scale bar equals 100 µm. ns, not significant.
    Figure Legend Snippet: Expression of SARS-CoV-2 entry factors in BRON and ALV organoids. (A) ACE2 (left) and TMPRSS2 (right) expression is detected in patient L2 organoids of bronchiolar (BRON) and alveolar (ALV) differentiation from passage 1-4. Relative quantification was performed using the Livac method and alveolar organoids are normalized to the average delta Ct of bronchiolar organoids. The mean value of three technical replicates for each passage are shown as individual data points. GraphPadPrism v9 was used to calculate and display the geometric mean and 90% confidence interval of the biological replicates (passages). Statistics were performed on the delta Ct values using the Mann-Whitney test. No statistically significant differences were observed in the median deltaCt values of ACE2 between BRON and ALV cultures. (B) Immunofluorescent staining of formalin fixed paraffin embedded (FFPE) sections of bronchiolar (BRON) organoids (representative images from patient L2 passage 2), display co-staining of the cilia marker (ARL13B) and the SARS-CoV-2 receptor ACE2 (transparent arrowheads). ACE2 staining alone (white arrowheads). Scale bar equals 100 µm. (C) Immunofluorescent staining of formalin fixed paraffin embedded (FFPE) sections of alveolar (ALV) organoids (representative images from patient L2 passage 2), display co-staining of the cilia marker (ARL13B) and the SARS-CoV-2 receptor ACE2 (transparent arrowheads). Scale bar equals 100 µm. ns, not significant.

    Techniques Used: Expressing, MANN-WHITNEY, Staining, Formalin-fixed Paraffin-Embedded, Marker

    Product information for TaqMan probes used in RT-qPCR assays.
    Figure Legend Snippet: Product information for TaqMan probes used in RT-qPCR assays.

    Techniques Used: Gene Assay

    The product information and dilution of primary used to detect the various target epitopes in this study.
    Figure Legend Snippet: The product information and dilution of primary used to detect the various target epitopes in this study.

    Techniques Used: Luciferase

    mouse monoclonal anti ace2 ab  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc mouse monoclonal anti ace2 ab
    SDS-PAGE analysis of maltose binding protein-angiotensin converting enzyme 2 N-terminal domain (MBP-ACE2NTD) fusion. (A) A schematic diagram of the fusion protein. (B) Total cellular proteins from IPTG-induced cells (NEB Express) containing the MBP-ACE2NTD fusion. Nine out of 11 clones tested here expressed the fusion (except #1 and #4). Clone #8 was chosen for further study. (C) MBP-ACE2NTD fusion expressed from IPTG induced cells (T7 Shuffle, lanes 1–4). Lane 1, total cellular protein; lane 2, flow-through from an amylose column; lane 3, eluted MBP-ACE2NTD protein from an amylose column; lane 4, MBP-ACE2NTD protein found in the pellet (inclusion body); lane 5, refolded MBP-ACE2NTD fusion protein from cell pellet (NEB Express); M, protein size marker (10–200 kDa, NEB). Arrows indicate the MBP-ACE2NTD protein, a truncated protein, and host MBP. (D) Purified MBP-ACE2NTD protein by amylose magnetic beads. Lane 1, refolded fusion; lanes 2 and 3, protein eluted in a Tris–HCl buffer (20 mM, pH 7.5) and sodium phosphate buffer (0.1 M, pH 8.0) with 10 mM maltose.
    Mouse Monoclonal Anti Ace2 Ab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A"

    Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2021.660149

    SDS-PAGE analysis of maltose binding protein-angiotensin converting enzyme 2 N-terminal domain (MBP-ACE2NTD) fusion. (A) A schematic diagram of the fusion protein. (B) Total cellular proteins from IPTG-induced cells (NEB Express) containing the MBP-ACE2NTD fusion. Nine out of 11 clones tested here expressed the fusion (except #1 and #4). Clone #8 was chosen for further study. (C) MBP-ACE2NTD fusion expressed from IPTG induced cells (T7 Shuffle, lanes 1–4). Lane 1, total cellular protein; lane 2, flow-through from an amylose column; lane 3, eluted MBP-ACE2NTD protein from an amylose column; lane 4, MBP-ACE2NTD protein found in the pellet (inclusion body); lane 5, refolded MBP-ACE2NTD fusion protein from cell pellet (NEB Express); M, protein size marker (10–200 kDa, NEB). Arrows indicate the MBP-ACE2NTD protein, a truncated protein, and host MBP. (D) Purified MBP-ACE2NTD protein by amylose magnetic beads. Lane 1, refolded fusion; lanes 2 and 3, protein eluted in a Tris–HCl buffer (20 mM, pH 7.5) and sodium phosphate buffer (0.1 M, pH 8.0) with 10 mM maltose.
    Figure Legend Snippet: SDS-PAGE analysis of maltose binding protein-angiotensin converting enzyme 2 N-terminal domain (MBP-ACE2NTD) fusion. (A) A schematic diagram of the fusion protein. (B) Total cellular proteins from IPTG-induced cells (NEB Express) containing the MBP-ACE2NTD fusion. Nine out of 11 clones tested here expressed the fusion (except #1 and #4). Clone #8 was chosen for further study. (C) MBP-ACE2NTD fusion expressed from IPTG induced cells (T7 Shuffle, lanes 1–4). Lane 1, total cellular protein; lane 2, flow-through from an amylose column; lane 3, eluted MBP-ACE2NTD protein from an amylose column; lane 4, MBP-ACE2NTD protein found in the pellet (inclusion body); lane 5, refolded MBP-ACE2NTD fusion protein from cell pellet (NEB Express); M, protein size marker (10–200 kDa, NEB). Arrows indicate the MBP-ACE2NTD protein, a truncated protein, and host MBP. (D) Purified MBP-ACE2NTD protein by amylose magnetic beads. Lane 1, refolded fusion; lanes 2 and 3, protein eluted in a Tris–HCl buffer (20 mM, pH 7.5) and sodium phosphate buffer (0.1 M, pH 8.0) with 10 mM maltose.

    Techniques Used: SDS Page, Binding Assay, Clone Assay, Marker, Purification, Magnetic Beads

    SDS-PAGE and Western blot analysis of RNase I-ACE2NTD fusion and activity assays. (A) Schematic diagram of RNase I-ACENTD (6×His) fusion. (B) Western blot analysis of RNase I-ACE2NTD in total protein, supernatant (soluble), and refolded protein using anti-His mAb. (C) Same as in (B) , except using anti-ACE2 mAb. (D) SDS-PAGE analysis of the refolded RNase I-ACE2NTD fusion and further purified protein by Ni magnetic beads and Ni spin column. (E) RNase I-ACE2NTD (refolded) ribonuclease activity on fluorescein (FL)-labeled RNA (300 nt) in NEB buffer 3. RNase I (6×His) and MBP-RNase I were used as positive controls. (F) Ribonuclease activity of RNase I-ACE2NTD (purified by Ni magnetic beads or Ni spin column) on SARS-CoV-2 RNA (50 mer). RNase I f , a positive control. FAM-S, FAM-labeled substrate; FAM-P, FAM labeled cleavage product(s).
    Figure Legend Snippet: SDS-PAGE and Western blot analysis of RNase I-ACE2NTD fusion and activity assays. (A) Schematic diagram of RNase I-ACENTD (6×His) fusion. (B) Western blot analysis of RNase I-ACE2NTD in total protein, supernatant (soluble), and refolded protein using anti-His mAb. (C) Same as in (B) , except using anti-ACE2 mAb. (D) SDS-PAGE analysis of the refolded RNase I-ACE2NTD fusion and further purified protein by Ni magnetic beads and Ni spin column. (E) RNase I-ACE2NTD (refolded) ribonuclease activity on fluorescein (FL)-labeled RNA (300 nt) in NEB buffer 3. RNase I (6×His) and MBP-RNase I were used as positive controls. (F) Ribonuclease activity of RNase I-ACE2NTD (purified by Ni magnetic beads or Ni spin column) on SARS-CoV-2 RNA (50 mer). RNase I f , a positive control. FAM-S, FAM-labeled substrate; FAM-P, FAM labeled cleavage product(s).

    Techniques Used: SDS Page, Western Blot, Activity Assay, Purification, Magnetic Beads, Labeling, Positive Control

    Expression of hRNase A-ACE2NTD150 in T7 Express LysY/ lacI q (C3013). (A) Schematic diagram of the fusion protein. (B) SDS-PAGE analysis of five isolates of hRNase A-ACE2NTD150 (five isolates with MBP signal peptide, five clones without MBP signal peptide). Total cell lysate of pET21b serves as a negative control. (C,D) Western blot analysis of the fusion proteins (soluble fraction/supernatant) by anti-His mAb and anti-ACE2 mAb.
    Figure Legend Snippet: Expression of hRNase A-ACE2NTD150 in T7 Express LysY/ lacI q (C3013). (A) Schematic diagram of the fusion protein. (B) SDS-PAGE analysis of five isolates of hRNase A-ACE2NTD150 (five isolates with MBP signal peptide, five clones without MBP signal peptide). Total cell lysate of pET21b serves as a negative control. (C,D) Western blot analysis of the fusion proteins (soluble fraction/supernatant) by anti-His mAb and anti-ACE2 mAb.

    Techniques Used: Expressing, SDS Page, Clone Assay, Negative Control, Western Blot

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  • 94
    Cell Signaling Technology Inc anti human ace2
    (A) Western blot analysis of endogenous <t>ACE2</t> in various types of cell lines, including HEK293T transfected with either control shRNAs (shCtrl) or shRNAs against ACE2 (shACE2), 2fTGH, Caco-2 and A549 cells. (B) HEK293T cells were transfected with shCtrl or increasing amounts of shACE2. Then cells were subjected to RT-qPCR analysis of Ace2 mRNA levels (right), or were infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) for 2 hrs. RT-qPCR was used to analyze SARS-CoV-2 RNA levels (left). (C) Western blot analysis of ACE2 in Caco-2 cells treated with vitamin (Vit) compounds (VitB1, 500 µM; VitB6, 500 µM; VitB12, 50 nM; VitC, 5 mM; VitD3, 25 µM; VitK1, 0.5 µM) for 24 hrs. (D) Western blot analysis of ACE2 in HEK293T, 2fTGH, Caco-2 and A549 cells treated with VitC at indicated concentrations for 24 hrs. (E) Western blot analysis of ACE2 in HEK293T cells treated with 5 mM or 0.2 mM of VitC for different durations. (F) Immunofluorescence analysis of ACE2 proteins in HeLa cells treated with VitC (5 mM) for 24 hrs. DAPI was used for the nucleus. Scale bars, 1 μm. (G) Western blot analysis of ACE2, IRF3 and STAT1 in A549 cells treated with VitC at indicated concentrations for 24 hrs. (H) Fluorescence microscopy of the SARS-CoV-2 GFP/ΔN or VSV-GFP viruses in Caco-2-N cells pretreated with VitC (5 mM and 10 mM) for 24 hrs, and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV-GFP (MOI = 0.1) viruses for 24 hrs. Scale bar: 100 µm. (I) RT-qPCR analysis of SARS-CoV-2 GFP/ΔN or VSV RNA levels in Caco-2 cells pretreated with VitC as (H), and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV (MOI = 0.1) for 2 hrs. Data are representative of three independent experiments (A, C-F), or are shown as mean and s.d. of three biological replicates (B, I). N.S, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S1.
    Anti Human Ace2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    94
    Cell Signaling Technology Inc anti ace2
    (A) Western blot analysis of endogenous <t>ACE2</t> in various types of cell lines, including HEK293T transfected with either control shRNAs (shCtrl) or shRNAs against ACE2 (shACE2), 2fTGH, Caco-2 and A549 cells. (B) HEK293T cells were transfected with shCtrl or increasing amounts of shACE2. Then cells were subjected to RT-qPCR analysis of Ace2 mRNA levels (right), or were infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) for 2 hrs. RT-qPCR was used to analyze SARS-CoV-2 RNA levels (left). (C) Western blot analysis of ACE2 in Caco-2 cells treated with vitamin (Vit) compounds (VitB1, 500 µM; VitB6, 500 µM; VitB12, 50 nM; VitC, 5 mM; VitD3, 25 µM; VitK1, 0.5 µM) for 24 hrs. (D) Western blot analysis of ACE2 in HEK293T, 2fTGH, Caco-2 and A549 cells treated with VitC at indicated concentrations for 24 hrs. (E) Western blot analysis of ACE2 in HEK293T cells treated with 5 mM or 0.2 mM of VitC for different durations. (F) Immunofluorescence analysis of ACE2 proteins in HeLa cells treated with VitC (5 mM) for 24 hrs. DAPI was used for the nucleus. Scale bars, 1 μm. (G) Western blot analysis of ACE2, IRF3 and STAT1 in A549 cells treated with VitC at indicated concentrations for 24 hrs. (H) Fluorescence microscopy of the SARS-CoV-2 GFP/ΔN or VSV-GFP viruses in Caco-2-N cells pretreated with VitC (5 mM and 10 mM) for 24 hrs, and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV-GFP (MOI = 0.1) viruses for 24 hrs. Scale bar: 100 µm. (I) RT-qPCR analysis of SARS-CoV-2 GFP/ΔN or VSV RNA levels in Caco-2 cells pretreated with VitC as (H), and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV (MOI = 0.1) for 2 hrs. Data are representative of three independent experiments (A, C-F), or are shown as mean and s.d. of three biological replicates (B, I). N.S, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S1.
    Anti Ace2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc ace2 oti1g4
    Expression of SARS-CoV-2 entry factors in BRON and ALV organoids. (A) <t>ACE2</t> (left) and TMPRSS2 (right) expression is detected in patient L2 organoids of bronchiolar (BRON) and alveolar (ALV) differentiation from passage 1-4. Relative quantification was performed using the Livac method and alveolar organoids are normalized to the average delta Ct of bronchiolar organoids. The mean value of three technical replicates for each passage are shown as individual data points. GraphPadPrism v9 was used to calculate and display the geometric mean and 90% confidence interval of the biological replicates (passages). Statistics were performed on the delta Ct values using the Mann-Whitney test. No statistically significant differences were observed in the median deltaCt values of ACE2 between BRON and ALV cultures. (B) Immunofluorescent staining of formalin fixed paraffin embedded (FFPE) sections of bronchiolar (BRON) organoids (representative images from patient L2 passage 2), display co-staining of the cilia marker (ARL13B) and the SARS-CoV-2 receptor ACE2 (transparent arrowheads). ACE2 staining alone (white arrowheads). Scale bar equals 100 µm. (C) Immunofluorescent staining of formalin fixed paraffin embedded (FFPE) sections of alveolar (ALV) organoids (representative images from patient L2 passage 2), display co-staining of the cilia marker (ARL13B) and the SARS-CoV-2 receptor ACE2 (transparent arrowheads). Scale bar equals 100 µm. ns, not significant.
    Ace2 Oti1g4, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc mouse monoclonal anti ace2 ab
    SDS-PAGE analysis of maltose binding protein-angiotensin converting enzyme 2 N-terminal domain (MBP-ACE2NTD) fusion. (A) A schematic diagram of the fusion protein. (B) Total cellular proteins from IPTG-induced cells (NEB Express) containing the MBP-ACE2NTD fusion. Nine out of 11 clones tested here expressed the fusion (except #1 and #4). Clone #8 was chosen for further study. (C) MBP-ACE2NTD fusion expressed from IPTG induced cells (T7 Shuffle, lanes 1–4). Lane 1, total cellular protein; lane 2, flow-through from an amylose column; lane 3, eluted MBP-ACE2NTD protein from an amylose column; lane 4, MBP-ACE2NTD protein found in the pellet (inclusion body); lane 5, refolded MBP-ACE2NTD fusion protein from cell pellet (NEB Express); M, protein size marker (10–200 kDa, NEB). Arrows indicate the MBP-ACE2NTD protein, a truncated protein, and host MBP. (D) Purified MBP-ACE2NTD protein by amylose magnetic beads. Lane 1, refolded fusion; lanes 2 and 3, protein eluted in a Tris–HCl buffer (20 mM, pH 7.5) and sodium phosphate buffer (0.1 M, pH 8.0) with 10 mM maltose.
    Mouse Monoclonal Anti Ace2 Ab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (A) Western blot analysis of endogenous ACE2 in various types of cell lines, including HEK293T transfected with either control shRNAs (shCtrl) or shRNAs against ACE2 (shACE2), 2fTGH, Caco-2 and A549 cells. (B) HEK293T cells were transfected with shCtrl or increasing amounts of shACE2. Then cells were subjected to RT-qPCR analysis of Ace2 mRNA levels (right), or were infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) for 2 hrs. RT-qPCR was used to analyze SARS-CoV-2 RNA levels (left). (C) Western blot analysis of ACE2 in Caco-2 cells treated with vitamin (Vit) compounds (VitB1, 500 µM; VitB6, 500 µM; VitB12, 50 nM; VitC, 5 mM; VitD3, 25 µM; VitK1, 0.5 µM) for 24 hrs. (D) Western blot analysis of ACE2 in HEK293T, 2fTGH, Caco-2 and A549 cells treated with VitC at indicated concentrations for 24 hrs. (E) Western blot analysis of ACE2 in HEK293T cells treated with 5 mM or 0.2 mM of VitC for different durations. (F) Immunofluorescence analysis of ACE2 proteins in HeLa cells treated with VitC (5 mM) for 24 hrs. DAPI was used for the nucleus. Scale bars, 1 μm. (G) Western blot analysis of ACE2, IRF3 and STAT1 in A549 cells treated with VitC at indicated concentrations for 24 hrs. (H) Fluorescence microscopy of the SARS-CoV-2 GFP/ΔN or VSV-GFP viruses in Caco-2-N cells pretreated with VitC (5 mM and 10 mM) for 24 hrs, and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV-GFP (MOI = 0.1) viruses for 24 hrs. Scale bar: 100 µm. (I) RT-qPCR analysis of SARS-CoV-2 GFP/ΔN or VSV RNA levels in Caco-2 cells pretreated with VitC as (H), and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV (MOI = 0.1) for 2 hrs. Data are representative of three independent experiments (A, C-F), or are shown as mean and s.d. of three biological replicates (B, I). N.S, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S1.

    Journal: bioRxiv

    Article Title: Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

    doi: 10.1101/2022.07.14.499651

    Figure Lengend Snippet: (A) Western blot analysis of endogenous ACE2 in various types of cell lines, including HEK293T transfected with either control shRNAs (shCtrl) or shRNAs against ACE2 (shACE2), 2fTGH, Caco-2 and A549 cells. (B) HEK293T cells were transfected with shCtrl or increasing amounts of shACE2. Then cells were subjected to RT-qPCR analysis of Ace2 mRNA levels (right), or were infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) for 2 hrs. RT-qPCR was used to analyze SARS-CoV-2 RNA levels (left). (C) Western blot analysis of ACE2 in Caco-2 cells treated with vitamin (Vit) compounds (VitB1, 500 µM; VitB6, 500 µM; VitB12, 50 nM; VitC, 5 mM; VitD3, 25 µM; VitK1, 0.5 µM) for 24 hrs. (D) Western blot analysis of ACE2 in HEK293T, 2fTGH, Caco-2 and A549 cells treated with VitC at indicated concentrations for 24 hrs. (E) Western blot analysis of ACE2 in HEK293T cells treated with 5 mM or 0.2 mM of VitC for different durations. (F) Immunofluorescence analysis of ACE2 proteins in HeLa cells treated with VitC (5 mM) for 24 hrs. DAPI was used for the nucleus. Scale bars, 1 μm. (G) Western blot analysis of ACE2, IRF3 and STAT1 in A549 cells treated with VitC at indicated concentrations for 24 hrs. (H) Fluorescence microscopy of the SARS-CoV-2 GFP/ΔN or VSV-GFP viruses in Caco-2-N cells pretreated with VitC (5 mM and 10 mM) for 24 hrs, and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV-GFP (MOI = 0.1) viruses for 24 hrs. Scale bar: 100 µm. (I) RT-qPCR analysis of SARS-CoV-2 GFP/ΔN or VSV RNA levels in Caco-2 cells pretreated with VitC as (H), and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV (MOI = 0.1) for 2 hrs. Data are representative of three independent experiments (A, C-F), or are shown as mean and s.d. of three biological replicates (B, I). N.S, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S1.

    Article Snippet: The antibodies with the indicated dilutions were as follows: anti-human ACE2 (Cell Signaling Technology, #74512, 1:1000), anti-ACE2 (Affinity, AF5165, 1:1000), anti-USP50 (Affinity, AF9225, 1:1000), anti-USP50 (Proteintech, 20374-1-AP, 1:1000), anti-Flag (Sigma, F7425, 1:5000), anti-HA (Abcam, ab9110, 1:3000), anti-Myc (Abmart, M20002H, 1:3000), anti-IRF3 (Santa Cruz, sc-33641, 1:1000), anti-STAT1 (Cell Signaling Technology, 9172, 1:1000), anti-Ubiquitin (Ub) (Santa Cruz, 12987-1-AP, 1:1000), anti-K48 Ub (Cell Signaling Technology, 4289S, 1:1000), anti-VSV-G (Santa Cruz, sc-66180, 1:2000) and anti-Tubulin (Proteintech, 66031-1-Ig, 1:3000).

    Techniques: Western Blot, Transfection, Quantitative RT-PCR, Infection, Immunofluorescence, Fluorescence, Microscopy, Two Tailed Test

    (A) RT-qPCR analysis of Ace2 mRNA in 2fTGH cells treated with VitC (5 mM) as indicated. (B) Western blot analysis of ACE2 in 2fTGH cells pretreated with ddH 2 O (Ctrl) or VitC (5 mM) for 12 hrs and then treated with CHX (50 μM) for 6 and 12 hrs. (C) Western blot analysis of Myc-ACE2 levels in HEK293T cells transfected with Myc-ACE2 and then treated with VitC at indicated concentrations for 12 hrs. (D) Western blot analysis of ACE2 in HEK293T cells pretreated with MG132 (10 µM) or MA (10 µM) for 2 hrs, followed by VitC treatment (5 mM) for 6 hrs. (E) Immunoprecipitation (IP)-immunoblotting (IB) analysis of ubiquitination (Ub) of endogenous ACE2 in 2fTGH cells treated with VitC at indicated concentrations for 12 hrs. (F) IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2 and different types of HA-Ub, and then treated with VitC (5 mM) for 12 hrs. (G) IP-IB analysis of K48-linked polyubiquitination (K48-Ub) of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 and then treated with VitC (2.5 mM and 5 mM) for 12 hrs, using a specific anti-K48-Ub antibody. (H) IP-IB analysis of K48-Ub of endogenous ACE2 in 2fTGH cells treated with VitC (2.5 mM and 5 mM) for 12 hrs. Data are representative of three independent experiments (B-H), or are shown as mean and s.d. of three biological replicates (A, B). N.S, not significant, ** p < 0.01 (two-tailed unpaired Student’s t -test). See also Figure S2.

    Journal: bioRxiv

    Article Title: Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

    doi: 10.1101/2022.07.14.499651

    Figure Lengend Snippet: (A) RT-qPCR analysis of Ace2 mRNA in 2fTGH cells treated with VitC (5 mM) as indicated. (B) Western blot analysis of ACE2 in 2fTGH cells pretreated with ddH 2 O (Ctrl) or VitC (5 mM) for 12 hrs and then treated with CHX (50 μM) for 6 and 12 hrs. (C) Western blot analysis of Myc-ACE2 levels in HEK293T cells transfected with Myc-ACE2 and then treated with VitC at indicated concentrations for 12 hrs. (D) Western blot analysis of ACE2 in HEK293T cells pretreated with MG132 (10 µM) or MA (10 µM) for 2 hrs, followed by VitC treatment (5 mM) for 6 hrs. (E) Immunoprecipitation (IP)-immunoblotting (IB) analysis of ubiquitination (Ub) of endogenous ACE2 in 2fTGH cells treated with VitC at indicated concentrations for 12 hrs. (F) IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2 and different types of HA-Ub, and then treated with VitC (5 mM) for 12 hrs. (G) IP-IB analysis of K48-linked polyubiquitination (K48-Ub) of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 and then treated with VitC (2.5 mM and 5 mM) for 12 hrs, using a specific anti-K48-Ub antibody. (H) IP-IB analysis of K48-Ub of endogenous ACE2 in 2fTGH cells treated with VitC (2.5 mM and 5 mM) for 12 hrs. Data are representative of three independent experiments (B-H), or are shown as mean and s.d. of three biological replicates (A, B). N.S, not significant, ** p < 0.01 (two-tailed unpaired Student’s t -test). See also Figure S2.

    Article Snippet: The antibodies with the indicated dilutions were as follows: anti-human ACE2 (Cell Signaling Technology, #74512, 1:1000), anti-ACE2 (Affinity, AF5165, 1:1000), anti-USP50 (Affinity, AF9225, 1:1000), anti-USP50 (Proteintech, 20374-1-AP, 1:1000), anti-Flag (Sigma, F7425, 1:5000), anti-HA (Abcam, ab9110, 1:3000), anti-Myc (Abmart, M20002H, 1:3000), anti-IRF3 (Santa Cruz, sc-33641, 1:1000), anti-STAT1 (Cell Signaling Technology, 9172, 1:1000), anti-Ubiquitin (Ub) (Santa Cruz, 12987-1-AP, 1:1000), anti-K48 Ub (Cell Signaling Technology, 4289S, 1:1000), anti-VSV-G (Santa Cruz, sc-66180, 1:2000) and anti-Tubulin (Proteintech, 66031-1-Ig, 1:3000).

    Techniques: Quantitative RT-PCR, Western Blot, Transfection, Immunoprecipitation, Two Tailed Test

    (A) Western blot analysis of ACE2 in HEK293T cells pretreated with PR619 (50 µM, 2 hrs) and then treated with VitC (5 mM) for 12 hrs. (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. (C) IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells cotransfected with these two constructs. (D) Immunoprecipitation analysis of the interaction between endogenous USP50 and ACE2 in 2fTGH cells. (E) Western blot analysis of ACE2 in HEK293T cells transfected with increasing amounts of Flag-USP50. (F) Western blot analysis of ACE2 in HEK293T cells transfected with shCtrl or shUSP50 (#1 or #2). (G) Western blot analysis of ACE2 in HEK293T cells transfected with Flag-USP50 and then treated with CHX (50 μM) as indicated. (H) Western blot analysis of ACE2 in Usp50 +/+ and Usp50 -/- HEK293T cells. (I) Western blot analysis of ACE2 in Usp50 +/+ and Usp50 -/- HEK293T cells treated with VitC (5 mM) for 12 hrs. (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 24 hrs. (K) Fluorescence microscopy of the SARS-CoV-2-S pseudovirus in Usp50 +/+ and Usp50 -/- HEK293T cells pretreated with or without VitC (5 mM) for 12 hrs, followed by infection with SARS-CoV-2-S pseudovirus (MOI = 0.1) for 24 hrs. Scale bar: 100 µm. Data are representative of three independent experiments (A-I), or are shown as mean and s.d. of three biological replicates (J, K). N.S, not significant, * p < 0.05, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S3.

    Journal: bioRxiv

    Article Title: Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

    doi: 10.1101/2022.07.14.499651

    Figure Lengend Snippet: (A) Western blot analysis of ACE2 in HEK293T cells pretreated with PR619 (50 µM, 2 hrs) and then treated with VitC (5 mM) for 12 hrs. (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. (C) IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells cotransfected with these two constructs. (D) Immunoprecipitation analysis of the interaction between endogenous USP50 and ACE2 in 2fTGH cells. (E) Western blot analysis of ACE2 in HEK293T cells transfected with increasing amounts of Flag-USP50. (F) Western blot analysis of ACE2 in HEK293T cells transfected with shCtrl or shUSP50 (#1 or #2). (G) Western blot analysis of ACE2 in HEK293T cells transfected with Flag-USP50 and then treated with CHX (50 μM) as indicated. (H) Western blot analysis of ACE2 in Usp50 +/+ and Usp50 -/- HEK293T cells. (I) Western blot analysis of ACE2 in Usp50 +/+ and Usp50 -/- HEK293T cells treated with VitC (5 mM) for 12 hrs. (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 24 hrs. (K) Fluorescence microscopy of the SARS-CoV-2-S pseudovirus in Usp50 +/+ and Usp50 -/- HEK293T cells pretreated with or without VitC (5 mM) for 12 hrs, followed by infection with SARS-CoV-2-S pseudovirus (MOI = 0.1) for 24 hrs. Scale bar: 100 µm. Data are representative of three independent experiments (A-I), or are shown as mean and s.d. of three biological replicates (J, K). N.S, not significant, * p < 0.05, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S3.

    Article Snippet: The antibodies with the indicated dilutions were as follows: anti-human ACE2 (Cell Signaling Technology, #74512, 1:1000), anti-ACE2 (Affinity, AF5165, 1:1000), anti-USP50 (Affinity, AF9225, 1:1000), anti-USP50 (Proteintech, 20374-1-AP, 1:1000), anti-Flag (Sigma, F7425, 1:5000), anti-HA (Abcam, ab9110, 1:3000), anti-Myc (Abmart, M20002H, 1:3000), anti-IRF3 (Santa Cruz, sc-33641, 1:1000), anti-STAT1 (Cell Signaling Technology, 9172, 1:1000), anti-Ubiquitin (Ub) (Santa Cruz, 12987-1-AP, 1:1000), anti-K48 Ub (Cell Signaling Technology, 4289S, 1:1000), anti-VSV-G (Santa Cruz, sc-66180, 1:2000) and anti-Tubulin (Proteintech, 66031-1-Ig, 1:3000).

    Techniques: Western Blot, Transfection, Expressing, Construct, Immunoprecipitation, Quantitative RT-PCR, Infection, Fluorescence, Microscopy, Two Tailed Test

    (A) IP-IB analysis of ubiquitination of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2, together with Flag-USP50 (WT) or its deubiquitinase inactive mutant (C53S). (B) IP-IB analysis of ubiquitination of endogenous ACE2 in HEK293T cells transfected with either shCtrl (-) or shUSP50 (#1, #2). (C) IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2, Flag-USP50 and different types of HA-Ub. (D) IP-IB analysis of K48-Ub of endogenous ACE2 in HEK293T cells transfected with shCtrl (-) or shUSP50 (#1, #2). (E) Putative ubiquitination sites of ACE2 in the PhosphoSitePlus database (Upper). Myc-ACE2 K48-linked ubiquitination was analyzed by IP-IB in HEK293T cells cotransfected with Myc-ACE2 (WT or its mutants) and HA-K48-Ub (Lower). (F) IP-IB analysis of Myc-ACE2 K48-linked ubiquitination in HEK293T cells cotransfected with Myc-ACE2 (WT or K788R) and HA-K48, together with shCtrl or shUSP50. (G) Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with CHX (50 μM) as indicated. (H) IP-IB analysis of K48-Ub of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) for 12 hrs, by a specific anti-K48-Ub antibody. (I) Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) as indicated. Data are representative of three independent experiments (A-I). See also Figure S3 and S4.

    Journal: bioRxiv

    Article Title: Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

    doi: 10.1101/2022.07.14.499651

    Figure Lengend Snippet: (A) IP-IB analysis of ubiquitination of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2, together with Flag-USP50 (WT) or its deubiquitinase inactive mutant (C53S). (B) IP-IB analysis of ubiquitination of endogenous ACE2 in HEK293T cells transfected with either shCtrl (-) or shUSP50 (#1, #2). (C) IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2, Flag-USP50 and different types of HA-Ub. (D) IP-IB analysis of K48-Ub of endogenous ACE2 in HEK293T cells transfected with shCtrl (-) or shUSP50 (#1, #2). (E) Putative ubiquitination sites of ACE2 in the PhosphoSitePlus database (Upper). Myc-ACE2 K48-linked ubiquitination was analyzed by IP-IB in HEK293T cells cotransfected with Myc-ACE2 (WT or its mutants) and HA-K48-Ub (Lower). (F) IP-IB analysis of Myc-ACE2 K48-linked ubiquitination in HEK293T cells cotransfected with Myc-ACE2 (WT or K788R) and HA-K48, together with shCtrl or shUSP50. (G) Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with CHX (50 μM) as indicated. (H) IP-IB analysis of K48-Ub of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) for 12 hrs, by a specific anti-K48-Ub antibody. (I) Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) as indicated. Data are representative of three independent experiments (A-I). See also Figure S3 and S4.

    Article Snippet: The antibodies with the indicated dilutions were as follows: anti-human ACE2 (Cell Signaling Technology, #74512, 1:1000), anti-ACE2 (Affinity, AF5165, 1:1000), anti-USP50 (Affinity, AF9225, 1:1000), anti-USP50 (Proteintech, 20374-1-AP, 1:1000), anti-Flag (Sigma, F7425, 1:5000), anti-HA (Abcam, ab9110, 1:3000), anti-Myc (Abmart, M20002H, 1:3000), anti-IRF3 (Santa Cruz, sc-33641, 1:1000), anti-STAT1 (Cell Signaling Technology, 9172, 1:1000), anti-Ubiquitin (Ub) (Santa Cruz, 12987-1-AP, 1:1000), anti-K48 Ub (Cell Signaling Technology, 4289S, 1:1000), anti-VSV-G (Santa Cruz, sc-66180, 1:2000) and anti-Tubulin (Proteintech, 66031-1-Ig, 1:3000).

    Techniques: Transfection, Mutagenesis, Western Blot

    (A) The procedure for analysis of the binding of VitC to Flag-USP50 or Myc-ACE2 (left) can be seen detailedly in the Methods. The concentrations of VitC that binds with either Flag-USP50 (middle) or Myc-ACE2 (right) proteins were measured by the Vitamin-C Detection Kit. (B) Recombinant human ACE2-IgG-Fc proteins (r-hACE2-Fc) or anti-Tyk2 IgG proteins were incubated with protein-G beads for 2 hrs, and then VitC was added for binding. After washing and centrifuging, the concentrations of VitC that binds to anti-Tyk2 proteins or r-hACE2-Fc proteins were detected as (A). (C) The concentrations of VitC that binds to Myc-ACE2 (WT) or its deletion mutants were detected as (A). (D) VitC docking to the binding pocket of ACE2 by standard precision (SP) using the Glide docking module in the Schrödinger molecular simulation software. (E) The docking diagram between amino acids of ACE2 and VitC based on the SP docking scoring function. (F) IP-IB analysis of the interaction between USP50 and Myc-ACE2 (WT) or its deletion mutants (Δ19-200, Δ201-400, Δ401-600 and Δ601-805) in HEK293T. (G) IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells transfected with these two constructs and then treated with VitC (2.5 mM and 5 mM) for 12 hrs. (H) IP-IB analysis of the in vivo interaction between endogenous USP50 and ACE2 in 2fTGH cells treated with VitC (5 mM) for 12 hrs. (I) Myc-ACE2 and Flag-USP50 proteins were immunoprecipitated from HEK293T cells transfected with either Myc-ACE2 or Flag-USP50. Flag-USP50 proteins were eluted by the Flag (M2) agarose. After washing, Flag-USP50 proteins were mixed with the Myc beads with Myc-ACE2, together with or without VitC (5 mM) for 2 hrs. After centrifuging, Flag-USP50 proteins interacting with Myc-ACE2 were analyzed by immunoblotting. (J) Flag-USP50 proteins were obtained as (I). Flag-USP50 and r-hACE2 proteins were mixed, together with increasing amounts of VitC or with VitC-Na + (20 mM). HCl is a pH control (pH = 4). After 2 hrs incubation, ACE2 proteins were analyzed by immunoblotting by a specific anti-ACE2 antibody. Data are representative of three independent experiments (F-J), or are shown as mean and s.d. of three biological replicates (A-C). N.S, not significant. *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S5.

    Journal: bioRxiv

    Article Title: Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

    doi: 10.1101/2022.07.14.499651

    Figure Lengend Snippet: (A) The procedure for analysis of the binding of VitC to Flag-USP50 or Myc-ACE2 (left) can be seen detailedly in the Methods. The concentrations of VitC that binds with either Flag-USP50 (middle) or Myc-ACE2 (right) proteins were measured by the Vitamin-C Detection Kit. (B) Recombinant human ACE2-IgG-Fc proteins (r-hACE2-Fc) or anti-Tyk2 IgG proteins were incubated with protein-G beads for 2 hrs, and then VitC was added for binding. After washing and centrifuging, the concentrations of VitC that binds to anti-Tyk2 proteins or r-hACE2-Fc proteins were detected as (A). (C) The concentrations of VitC that binds to Myc-ACE2 (WT) or its deletion mutants were detected as (A). (D) VitC docking to the binding pocket of ACE2 by standard precision (SP) using the Glide docking module in the Schrödinger molecular simulation software. (E) The docking diagram between amino acids of ACE2 and VitC based on the SP docking scoring function. (F) IP-IB analysis of the interaction between USP50 and Myc-ACE2 (WT) or its deletion mutants (Δ19-200, Δ201-400, Δ401-600 and Δ601-805) in HEK293T. (G) IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells transfected with these two constructs and then treated with VitC (2.5 mM and 5 mM) for 12 hrs. (H) IP-IB analysis of the in vivo interaction between endogenous USP50 and ACE2 in 2fTGH cells treated with VitC (5 mM) for 12 hrs. (I) Myc-ACE2 and Flag-USP50 proteins were immunoprecipitated from HEK293T cells transfected with either Myc-ACE2 or Flag-USP50. Flag-USP50 proteins were eluted by the Flag (M2) agarose. After washing, Flag-USP50 proteins were mixed with the Myc beads with Myc-ACE2, together with or without VitC (5 mM) for 2 hrs. After centrifuging, Flag-USP50 proteins interacting with Myc-ACE2 were analyzed by immunoblotting. (J) Flag-USP50 proteins were obtained as (I). Flag-USP50 and r-hACE2 proteins were mixed, together with increasing amounts of VitC or with VitC-Na + (20 mM). HCl is a pH control (pH = 4). After 2 hrs incubation, ACE2 proteins were analyzed by immunoblotting by a specific anti-ACE2 antibody. Data are representative of three independent experiments (F-J), or are shown as mean and s.d. of three biological replicates (A-C). N.S, not significant. *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S5.

    Article Snippet: The antibodies with the indicated dilutions were as follows: anti-human ACE2 (Cell Signaling Technology, #74512, 1:1000), anti-ACE2 (Affinity, AF5165, 1:1000), anti-USP50 (Affinity, AF9225, 1:1000), anti-USP50 (Proteintech, 20374-1-AP, 1:1000), anti-Flag (Sigma, F7425, 1:5000), anti-HA (Abcam, ab9110, 1:3000), anti-Myc (Abmart, M20002H, 1:3000), anti-IRF3 (Santa Cruz, sc-33641, 1:1000), anti-STAT1 (Cell Signaling Technology, 9172, 1:1000), anti-Ubiquitin (Ub) (Santa Cruz, 12987-1-AP, 1:1000), anti-K48 Ub (Cell Signaling Technology, 4289S, 1:1000), anti-VSV-G (Santa Cruz, sc-66180, 1:2000) and anti-Tubulin (Proteintech, 66031-1-Ig, 1:3000).

    Techniques: Binding Assay, Recombinant, Incubation, Software, Transfection, Construct, In Vivo, Immunoprecipitation, Western Blot, Two Tailed Test

    (A) IP-IB analysis of the interaction between endogenous hACE2 and USP50 in the lung and liver tissues of hACE2 mice. (B) The hACE2 mice were intraperitoneally administrated with VitC (300 mg/day/kg body weight) for two days. The interaction between USP50 and hACE2 in mouse lung tissues was analyzed by IP-IB. (C) IP-IB analysis of K48-Ub of hACE2 in mouse lung tissues from (B). (D) Western blot analysis of hACE2 levels in lung tissues of hACE2 mice administrated with VitC as (B). (E) Immunohistochemical staining of hACE2 protein in the lung, kidney and liver tissues from (B). (F) The hACE2 mice were administrated with VitC as (B). Mice were then given intraperitoneal injections of SARS-CoV-2-S pseudoviruses (1×10 6 PFU per gram body). After 24 hrs, immunohistochemical staining was performed to analyze the SARS-CoV-2 Spike proteins in mouse lung and kidney tissues. Scale bar: 100 µm. (G) RT-qPCR analysis of the SARS-CoV-2 Spike mRNA levels in lung, kidney, liver and spleen tissues of hACE2 mice treated with VitC and SARS-CoV-2-S pseudoviruses as (F). Data are representative of three independent experiments (A-D). All graphs show the mean ± SEM for five individual mice (G). *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S6 and S7.

    Journal: bioRxiv

    Article Title: Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

    doi: 10.1101/2022.07.14.499651

    Figure Lengend Snippet: (A) IP-IB analysis of the interaction between endogenous hACE2 and USP50 in the lung and liver tissues of hACE2 mice. (B) The hACE2 mice were intraperitoneally administrated with VitC (300 mg/day/kg body weight) for two days. The interaction between USP50 and hACE2 in mouse lung tissues was analyzed by IP-IB. (C) IP-IB analysis of K48-Ub of hACE2 in mouse lung tissues from (B). (D) Western blot analysis of hACE2 levels in lung tissues of hACE2 mice administrated with VitC as (B). (E) Immunohistochemical staining of hACE2 protein in the lung, kidney and liver tissues from (B). (F) The hACE2 mice were administrated with VitC as (B). Mice were then given intraperitoneal injections of SARS-CoV-2-S pseudoviruses (1×10 6 PFU per gram body). After 24 hrs, immunohistochemical staining was performed to analyze the SARS-CoV-2 Spike proteins in mouse lung and kidney tissues. Scale bar: 100 µm. (G) RT-qPCR analysis of the SARS-CoV-2 Spike mRNA levels in lung, kidney, liver and spleen tissues of hACE2 mice treated with VitC and SARS-CoV-2-S pseudoviruses as (F). Data are representative of three independent experiments (A-D). All graphs show the mean ± SEM for five individual mice (G). *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S6 and S7.

    Article Snippet: The antibodies with the indicated dilutions were as follows: anti-human ACE2 (Cell Signaling Technology, #74512, 1:1000), anti-ACE2 (Affinity, AF5165, 1:1000), anti-USP50 (Affinity, AF9225, 1:1000), anti-USP50 (Proteintech, 20374-1-AP, 1:1000), anti-Flag (Sigma, F7425, 1:5000), anti-HA (Abcam, ab9110, 1:3000), anti-Myc (Abmart, M20002H, 1:3000), anti-IRF3 (Santa Cruz, sc-33641, 1:1000), anti-STAT1 (Cell Signaling Technology, 9172, 1:1000), anti-Ubiquitin (Ub) (Santa Cruz, 12987-1-AP, 1:1000), anti-K48 Ub (Cell Signaling Technology, 4289S, 1:1000), anti-VSV-G (Santa Cruz, sc-66180, 1:2000) and anti-Tubulin (Proteintech, 66031-1-Ig, 1:3000).

    Techniques: Western Blot, Immunohistochemical staining, Staining, Quantitative RT-PCR, Two Tailed Test

    (A) Western blot analysis of endogenous ACE2 in various types of cell lines, including HEK293T transfected with either control shRNAs (shCtrl) or shRNAs against ACE2 (shACE2), 2fTGH, Caco-2 and A549 cells. (B) HEK293T cells were transfected with shCtrl or increasing amounts of shACE2. Then cells were subjected to RT-qPCR analysis of Ace2 mRNA levels (right), or were infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) for 2 hrs. RT-qPCR was used to analyze SARS-CoV-2 RNA levels (left). (C) Western blot analysis of ACE2 in Caco-2 cells treated with vitamin (Vit) compounds (VitB1, 500 µM; VitB6, 500 µM; VitB12, 50 nM; VitC, 5 mM; VitD3, 25 µM; VitK1, 0.5 µM) for 24 hrs. (D) Western blot analysis of ACE2 in HEK293T, 2fTGH, Caco-2 and A549 cells treated with VitC at indicated concentrations for 24 hrs. (E) Western blot analysis of ACE2 in HEK293T cells treated with 5 mM or 0.2 mM of VitC for different durations. (F) Immunofluorescence analysis of ACE2 proteins in HeLa cells treated with VitC (5 mM) for 24 hrs. DAPI was used for the nucleus. Scale bars, 1 μm. (G) Western blot analysis of ACE2, IRF3 and STAT1 in A549 cells treated with VitC at indicated concentrations for 24 hrs. (H) Fluorescence microscopy of the SARS-CoV-2 GFP/ΔN or VSV-GFP viruses in Caco-2-N cells pretreated with VitC (5 mM and 10 mM) for 24 hrs, and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV-GFP (MOI = 0.1) viruses for 24 hrs. Scale bar: 100 µm. (I) RT-qPCR analysis of SARS-CoV-2 GFP/ΔN or VSV RNA levels in Caco-2 cells pretreated with VitC as (H), and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV (MOI = 0.1) for 2 hrs. Data are representative of three independent experiments (A, C-F), or are shown as mean and s.d. of three biological replicates (B, I). N.S, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S1.

    Journal: bioRxiv

    Article Title: Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

    doi: 10.1101/2022.07.14.499651

    Figure Lengend Snippet: (A) Western blot analysis of endogenous ACE2 in various types of cell lines, including HEK293T transfected with either control shRNAs (shCtrl) or shRNAs against ACE2 (shACE2), 2fTGH, Caco-2 and A549 cells. (B) HEK293T cells were transfected with shCtrl or increasing amounts of shACE2. Then cells were subjected to RT-qPCR analysis of Ace2 mRNA levels (right), or were infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) for 2 hrs. RT-qPCR was used to analyze SARS-CoV-2 RNA levels (left). (C) Western blot analysis of ACE2 in Caco-2 cells treated with vitamin (Vit) compounds (VitB1, 500 µM; VitB6, 500 µM; VitB12, 50 nM; VitC, 5 mM; VitD3, 25 µM; VitK1, 0.5 µM) for 24 hrs. (D) Western blot analysis of ACE2 in HEK293T, 2fTGH, Caco-2 and A549 cells treated with VitC at indicated concentrations for 24 hrs. (E) Western blot analysis of ACE2 in HEK293T cells treated with 5 mM or 0.2 mM of VitC for different durations. (F) Immunofluorescence analysis of ACE2 proteins in HeLa cells treated with VitC (5 mM) for 24 hrs. DAPI was used for the nucleus. Scale bars, 1 μm. (G) Western blot analysis of ACE2, IRF3 and STAT1 in A549 cells treated with VitC at indicated concentrations for 24 hrs. (H) Fluorescence microscopy of the SARS-CoV-2 GFP/ΔN or VSV-GFP viruses in Caco-2-N cells pretreated with VitC (5 mM and 10 mM) for 24 hrs, and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV-GFP (MOI = 0.1) viruses for 24 hrs. Scale bar: 100 µm. (I) RT-qPCR analysis of SARS-CoV-2 GFP/ΔN or VSV RNA levels in Caco-2 cells pretreated with VitC as (H), and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV (MOI = 0.1) for 2 hrs. Data are representative of three independent experiments (A, C-F), or are shown as mean and s.d. of three biological replicates (B, I). N.S, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S1.

    Article Snippet: The antibodies with the indicated dilutions were as follows: anti-human ACE2 (Cell Signaling Technology, #74512, 1:1000), anti-ACE2 (Affinity, AF5165, 1:1000), anti-USP50 (Affinity, AF9225, 1:1000), anti-USP50 (Proteintech, 20374-1-AP, 1:1000), anti-Flag (Sigma, F7425, 1:5000), anti-HA (Abcam, ab9110, 1:3000), anti-Myc (Abmart, M20002H, 1:3000), anti-IRF3 (Santa Cruz, sc-33641, 1:1000), anti-STAT1 (Cell Signaling Technology, 9172, 1:1000), anti-Ubiquitin (Ub) (Santa Cruz, 12987-1-AP, 1:1000), anti-K48 Ub (Cell Signaling Technology, 4289S, 1:1000), anti-VSV-G (Santa Cruz, sc-66180, 1:2000) and anti-Tubulin (Proteintech, 66031-1-Ig, 1:3000).

    Techniques: Western Blot, Transfection, Quantitative RT-PCR, Infection, Immunofluorescence, Fluorescence, Microscopy, Two Tailed Test

    (A) RT-qPCR analysis of Ace2 mRNA in 2fTGH cells treated with VitC (5 mM) as indicated. (B) Western blot analysis of ACE2 in 2fTGH cells pretreated with ddH 2 O (Ctrl) or VitC (5 mM) for 12 hrs and then treated with CHX (50 μM) for 6 and 12 hrs. (C) Western blot analysis of Myc-ACE2 levels in HEK293T cells transfected with Myc-ACE2 and then treated with VitC at indicated concentrations for 12 hrs. (D) Western blot analysis of ACE2 in HEK293T cells pretreated with MG132 (10 µM) or MA (10 µM) for 2 hrs, followed by VitC treatment (5 mM) for 6 hrs. (E) Immunoprecipitation (IP)-immunoblotting (IB) analysis of ubiquitination (Ub) of endogenous ACE2 in 2fTGH cells treated with VitC at indicated concentrations for 12 hrs. (F) IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2 and different types of HA-Ub, and then treated with VitC (5 mM) for 12 hrs. (G) IP-IB analysis of K48-linked polyubiquitination (K48-Ub) of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 and then treated with VitC (2.5 mM and 5 mM) for 12 hrs, using a specific anti-K48-Ub antibody. (H) IP-IB analysis of K48-Ub of endogenous ACE2 in 2fTGH cells treated with VitC (2.5 mM and 5 mM) for 12 hrs. Data are representative of three independent experiments (B-H), or are shown as mean and s.d. of three biological replicates (A, B). N.S, not significant, ** p < 0.01 (two-tailed unpaired Student’s t -test). See also Figure S2.

    Journal: bioRxiv

    Article Title: Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

    doi: 10.1101/2022.07.14.499651

    Figure Lengend Snippet: (A) RT-qPCR analysis of Ace2 mRNA in 2fTGH cells treated with VitC (5 mM) as indicated. (B) Western blot analysis of ACE2 in 2fTGH cells pretreated with ddH 2 O (Ctrl) or VitC (5 mM) for 12 hrs and then treated with CHX (50 μM) for 6 and 12 hrs. (C) Western blot analysis of Myc-ACE2 levels in HEK293T cells transfected with Myc-ACE2 and then treated with VitC at indicated concentrations for 12 hrs. (D) Western blot analysis of ACE2 in HEK293T cells pretreated with MG132 (10 µM) or MA (10 µM) for 2 hrs, followed by VitC treatment (5 mM) for 6 hrs. (E) Immunoprecipitation (IP)-immunoblotting (IB) analysis of ubiquitination (Ub) of endogenous ACE2 in 2fTGH cells treated with VitC at indicated concentrations for 12 hrs. (F) IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2 and different types of HA-Ub, and then treated with VitC (5 mM) for 12 hrs. (G) IP-IB analysis of K48-linked polyubiquitination (K48-Ub) of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 and then treated with VitC (2.5 mM and 5 mM) for 12 hrs, using a specific anti-K48-Ub antibody. (H) IP-IB analysis of K48-Ub of endogenous ACE2 in 2fTGH cells treated with VitC (2.5 mM and 5 mM) for 12 hrs. Data are representative of three independent experiments (B-H), or are shown as mean and s.d. of three biological replicates (A, B). N.S, not significant, ** p < 0.01 (two-tailed unpaired Student’s t -test). See also Figure S2.

    Article Snippet: The antibodies with the indicated dilutions were as follows: anti-human ACE2 (Cell Signaling Technology, #74512, 1:1000), anti-ACE2 (Affinity, AF5165, 1:1000), anti-USP50 (Affinity, AF9225, 1:1000), anti-USP50 (Proteintech, 20374-1-AP, 1:1000), anti-Flag (Sigma, F7425, 1:5000), anti-HA (Abcam, ab9110, 1:3000), anti-Myc (Abmart, M20002H, 1:3000), anti-IRF3 (Santa Cruz, sc-33641, 1:1000), anti-STAT1 (Cell Signaling Technology, 9172, 1:1000), anti-Ubiquitin (Ub) (Santa Cruz, 12987-1-AP, 1:1000), anti-K48 Ub (Cell Signaling Technology, 4289S, 1:1000), anti-VSV-G (Santa Cruz, sc-66180, 1:2000) and anti-Tubulin (Proteintech, 66031-1-Ig, 1:3000).

    Techniques: Quantitative RT-PCR, Western Blot, Transfection, Immunoprecipitation, Two Tailed Test

    (A) Western blot analysis of ACE2 in HEK293T cells pretreated with PR619 (50 µM, 2 hrs) and then treated with VitC (5 mM) for 12 hrs. (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. (C) IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells cotransfected with these two constructs. (D) Immunoprecipitation analysis of the interaction between endogenous USP50 and ACE2 in 2fTGH cells. (E) Western blot analysis of ACE2 in HEK293T cells transfected with increasing amounts of Flag-USP50. (F) Western blot analysis of ACE2 in HEK293T cells transfected with shCtrl or shUSP50 (#1 or #2). (G) Western blot analysis of ACE2 in HEK293T cells transfected with Flag-USP50 and then treated with CHX (50 μM) as indicated. (H) Western blot analysis of ACE2 in Usp50 +/+ and Usp50 -/- HEK293T cells. (I) Western blot analysis of ACE2 in Usp50 +/+ and Usp50 -/- HEK293T cells treated with VitC (5 mM) for 12 hrs. (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 24 hrs. (K) Fluorescence microscopy of the SARS-CoV-2-S pseudovirus in Usp50 +/+ and Usp50 -/- HEK293T cells pretreated with or without VitC (5 mM) for 12 hrs, followed by infection with SARS-CoV-2-S pseudovirus (MOI = 0.1) for 24 hrs. Scale bar: 100 µm. Data are representative of three independent experiments (A-I), or are shown as mean and s.d. of three biological replicates (J, K). N.S, not significant, * p < 0.05, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S3.

    Journal: bioRxiv

    Article Title: Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

    doi: 10.1101/2022.07.14.499651

    Figure Lengend Snippet: (A) Western blot analysis of ACE2 in HEK293T cells pretreated with PR619 (50 µM, 2 hrs) and then treated with VitC (5 mM) for 12 hrs. (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. (C) IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells cotransfected with these two constructs. (D) Immunoprecipitation analysis of the interaction between endogenous USP50 and ACE2 in 2fTGH cells. (E) Western blot analysis of ACE2 in HEK293T cells transfected with increasing amounts of Flag-USP50. (F) Western blot analysis of ACE2 in HEK293T cells transfected with shCtrl or shUSP50 (#1 or #2). (G) Western blot analysis of ACE2 in HEK293T cells transfected with Flag-USP50 and then treated with CHX (50 μM) as indicated. (H) Western blot analysis of ACE2 in Usp50 +/+ and Usp50 -/- HEK293T cells. (I) Western blot analysis of ACE2 in Usp50 +/+ and Usp50 -/- HEK293T cells treated with VitC (5 mM) for 12 hrs. (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 24 hrs. (K) Fluorescence microscopy of the SARS-CoV-2-S pseudovirus in Usp50 +/+ and Usp50 -/- HEK293T cells pretreated with or without VitC (5 mM) for 12 hrs, followed by infection with SARS-CoV-2-S pseudovirus (MOI = 0.1) for 24 hrs. Scale bar: 100 µm. Data are representative of three independent experiments (A-I), or are shown as mean and s.d. of three biological replicates (J, K). N.S, not significant, * p < 0.05, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S3.

    Article Snippet: The antibodies with the indicated dilutions were as follows: anti-human ACE2 (Cell Signaling Technology, #74512, 1:1000), anti-ACE2 (Affinity, AF5165, 1:1000), anti-USP50 (Affinity, AF9225, 1:1000), anti-USP50 (Proteintech, 20374-1-AP, 1:1000), anti-Flag (Sigma, F7425, 1:5000), anti-HA (Abcam, ab9110, 1:3000), anti-Myc (Abmart, M20002H, 1:3000), anti-IRF3 (Santa Cruz, sc-33641, 1:1000), anti-STAT1 (Cell Signaling Technology, 9172, 1:1000), anti-Ubiquitin (Ub) (Santa Cruz, 12987-1-AP, 1:1000), anti-K48 Ub (Cell Signaling Technology, 4289S, 1:1000), anti-VSV-G (Santa Cruz, sc-66180, 1:2000) and anti-Tubulin (Proteintech, 66031-1-Ig, 1:3000).

    Techniques: Western Blot, Transfection, Expressing, Construct, Immunoprecipitation, Quantitative RT-PCR, Infection, Fluorescence, Microscopy, Two Tailed Test

    (A) IP-IB analysis of ubiquitination of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2, together with Flag-USP50 (WT) or its deubiquitinase inactive mutant (C53S). (B) IP-IB analysis of ubiquitination of endogenous ACE2 in HEK293T cells transfected with either shCtrl (-) or shUSP50 (#1, #2). (C) IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2, Flag-USP50 and different types of HA-Ub. (D) IP-IB analysis of K48-Ub of endogenous ACE2 in HEK293T cells transfected with shCtrl (-) or shUSP50 (#1, #2). (E) Putative ubiquitination sites of ACE2 in the PhosphoSitePlus database (Upper). Myc-ACE2 K48-linked ubiquitination was analyzed by IP-IB in HEK293T cells cotransfected with Myc-ACE2 (WT or its mutants) and HA-K48-Ub (Lower). (F) IP-IB analysis of Myc-ACE2 K48-linked ubiquitination in HEK293T cells cotransfected with Myc-ACE2 (WT or K788R) and HA-K48, together with shCtrl or shUSP50. (G) Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with CHX (50 μM) as indicated. (H) IP-IB analysis of K48-Ub of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) for 12 hrs, by a specific anti-K48-Ub antibody. (I) Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) as indicated. Data are representative of three independent experiments (A-I). See also Figure S3 and S4.

    Journal: bioRxiv

    Article Title: Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

    doi: 10.1101/2022.07.14.499651

    Figure Lengend Snippet: (A) IP-IB analysis of ubiquitination of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2, together with Flag-USP50 (WT) or its deubiquitinase inactive mutant (C53S). (B) IP-IB analysis of ubiquitination of endogenous ACE2 in HEK293T cells transfected with either shCtrl (-) or shUSP50 (#1, #2). (C) IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2, Flag-USP50 and different types of HA-Ub. (D) IP-IB analysis of K48-Ub of endogenous ACE2 in HEK293T cells transfected with shCtrl (-) or shUSP50 (#1, #2). (E) Putative ubiquitination sites of ACE2 in the PhosphoSitePlus database (Upper). Myc-ACE2 K48-linked ubiquitination was analyzed by IP-IB in HEK293T cells cotransfected with Myc-ACE2 (WT or its mutants) and HA-K48-Ub (Lower). (F) IP-IB analysis of Myc-ACE2 K48-linked ubiquitination in HEK293T cells cotransfected with Myc-ACE2 (WT or K788R) and HA-K48, together with shCtrl or shUSP50. (G) Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with CHX (50 μM) as indicated. (H) IP-IB analysis of K48-Ub of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) for 12 hrs, by a specific anti-K48-Ub antibody. (I) Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) as indicated. Data are representative of three independent experiments (A-I). See also Figure S3 and S4.

    Article Snippet: The antibodies with the indicated dilutions were as follows: anti-human ACE2 (Cell Signaling Technology, #74512, 1:1000), anti-ACE2 (Affinity, AF5165, 1:1000), anti-USP50 (Affinity, AF9225, 1:1000), anti-USP50 (Proteintech, 20374-1-AP, 1:1000), anti-Flag (Sigma, F7425, 1:5000), anti-HA (Abcam, ab9110, 1:3000), anti-Myc (Abmart, M20002H, 1:3000), anti-IRF3 (Santa Cruz, sc-33641, 1:1000), anti-STAT1 (Cell Signaling Technology, 9172, 1:1000), anti-Ubiquitin (Ub) (Santa Cruz, 12987-1-AP, 1:1000), anti-K48 Ub (Cell Signaling Technology, 4289S, 1:1000), anti-VSV-G (Santa Cruz, sc-66180, 1:2000) and anti-Tubulin (Proteintech, 66031-1-Ig, 1:3000).

    Techniques: Transfection, Mutagenesis, Western Blot

    (A) The procedure for analysis of the binding of VitC to Flag-USP50 or Myc-ACE2 (left) can be seen detailedly in the Methods. The concentrations of VitC that binds with either Flag-USP50 (middle) or Myc-ACE2 (right) proteins were measured by the Vitamin-C Detection Kit. (B) Recombinant human ACE2-IgG-Fc proteins (r-hACE2-Fc) or anti-Tyk2 IgG proteins were incubated with protein-G beads for 2 hrs, and then VitC was added for binding. After washing and centrifuging, the concentrations of VitC that binds to anti-Tyk2 proteins or r-hACE2-Fc proteins were detected as (A). (C) The concentrations of VitC that binds to Myc-ACE2 (WT) or its deletion mutants were detected as (A). (D) VitC docking to the binding pocket of ACE2 by standard precision (SP) using the Glide docking module in the Schrödinger molecular simulation software. (E) The docking diagram between amino acids of ACE2 and VitC based on the SP docking scoring function. (F) IP-IB analysis of the interaction between USP50 and Myc-ACE2 (WT) or its deletion mutants (Δ19-200, Δ201-400, Δ401-600 and Δ601-805) in HEK293T. (G) IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells transfected with these two constructs and then treated with VitC (2.5 mM and 5 mM) for 12 hrs. (H) IP-IB analysis of the in vivo interaction between endogenous USP50 and ACE2 in 2fTGH cells treated with VitC (5 mM) for 12 hrs. (I) Myc-ACE2 and Flag-USP50 proteins were immunoprecipitated from HEK293T cells transfected with either Myc-ACE2 or Flag-USP50. Flag-USP50 proteins were eluted by the Flag (M2) agarose. After washing, Flag-USP50 proteins were mixed with the Myc beads with Myc-ACE2, together with or without VitC (5 mM) for 2 hrs. After centrifuging, Flag-USP50 proteins interacting with Myc-ACE2 were analyzed by immunoblotting. (J) Flag-USP50 proteins were obtained as (I). Flag-USP50 and r-hACE2 proteins were mixed, together with increasing amounts of VitC or with VitC-Na + (20 mM). HCl is a pH control (pH = 4). After 2 hrs incubation, ACE2 proteins were analyzed by immunoblotting by a specific anti-ACE2 antibody. Data are representative of three independent experiments (F-J), or are shown as mean and s.d. of three biological replicates (A-C). N.S, not significant. *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S5.

    Journal: bioRxiv

    Article Title: Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

    doi: 10.1101/2022.07.14.499651

    Figure Lengend Snippet: (A) The procedure for analysis of the binding of VitC to Flag-USP50 or Myc-ACE2 (left) can be seen detailedly in the Methods. The concentrations of VitC that binds with either Flag-USP50 (middle) or Myc-ACE2 (right) proteins were measured by the Vitamin-C Detection Kit. (B) Recombinant human ACE2-IgG-Fc proteins (r-hACE2-Fc) or anti-Tyk2 IgG proteins were incubated with protein-G beads for 2 hrs, and then VitC was added for binding. After washing and centrifuging, the concentrations of VitC that binds to anti-Tyk2 proteins or r-hACE2-Fc proteins were detected as (A). (C) The concentrations of VitC that binds to Myc-ACE2 (WT) or its deletion mutants were detected as (A). (D) VitC docking to the binding pocket of ACE2 by standard precision (SP) using the Glide docking module in the Schrödinger molecular simulation software. (E) The docking diagram between amino acids of ACE2 and VitC based on the SP docking scoring function. (F) IP-IB analysis of the interaction between USP50 and Myc-ACE2 (WT) or its deletion mutants (Δ19-200, Δ201-400, Δ401-600 and Δ601-805) in HEK293T. (G) IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells transfected with these two constructs and then treated with VitC (2.5 mM and 5 mM) for 12 hrs. (H) IP-IB analysis of the in vivo interaction between endogenous USP50 and ACE2 in 2fTGH cells treated with VitC (5 mM) for 12 hrs. (I) Myc-ACE2 and Flag-USP50 proteins were immunoprecipitated from HEK293T cells transfected with either Myc-ACE2 or Flag-USP50. Flag-USP50 proteins were eluted by the Flag (M2) agarose. After washing, Flag-USP50 proteins were mixed with the Myc beads with Myc-ACE2, together with or without VitC (5 mM) for 2 hrs. After centrifuging, Flag-USP50 proteins interacting with Myc-ACE2 were analyzed by immunoblotting. (J) Flag-USP50 proteins were obtained as (I). Flag-USP50 and r-hACE2 proteins were mixed, together with increasing amounts of VitC or with VitC-Na + (20 mM). HCl is a pH control (pH = 4). After 2 hrs incubation, ACE2 proteins were analyzed by immunoblotting by a specific anti-ACE2 antibody. Data are representative of three independent experiments (F-J), or are shown as mean and s.d. of three biological replicates (A-C). N.S, not significant. *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S5.

    Article Snippet: The antibodies with the indicated dilutions were as follows: anti-human ACE2 (Cell Signaling Technology, #74512, 1:1000), anti-ACE2 (Affinity, AF5165, 1:1000), anti-USP50 (Affinity, AF9225, 1:1000), anti-USP50 (Proteintech, 20374-1-AP, 1:1000), anti-Flag (Sigma, F7425, 1:5000), anti-HA (Abcam, ab9110, 1:3000), anti-Myc (Abmart, M20002H, 1:3000), anti-IRF3 (Santa Cruz, sc-33641, 1:1000), anti-STAT1 (Cell Signaling Technology, 9172, 1:1000), anti-Ubiquitin (Ub) (Santa Cruz, 12987-1-AP, 1:1000), anti-K48 Ub (Cell Signaling Technology, 4289S, 1:1000), anti-VSV-G (Santa Cruz, sc-66180, 1:2000) and anti-Tubulin (Proteintech, 66031-1-Ig, 1:3000).

    Techniques: Binding Assay, Recombinant, Incubation, Software, Transfection, Construct, In Vivo, Immunoprecipitation, Western Blot, Two Tailed Test

    Expression of SARS-CoV-2 entry factors in BRON and ALV organoids. (A) ACE2 (left) and TMPRSS2 (right) expression is detected in patient L2 organoids of bronchiolar (BRON) and alveolar (ALV) differentiation from passage 1-4. Relative quantification was performed using the Livac method and alveolar organoids are normalized to the average delta Ct of bronchiolar organoids. The mean value of three technical replicates for each passage are shown as individual data points. GraphPadPrism v9 was used to calculate and display the geometric mean and 90% confidence interval of the biological replicates (passages). Statistics were performed on the delta Ct values using the Mann-Whitney test. No statistically significant differences were observed in the median deltaCt values of ACE2 between BRON and ALV cultures. (B) Immunofluorescent staining of formalin fixed paraffin embedded (FFPE) sections of bronchiolar (BRON) organoids (representative images from patient L2 passage 2), display co-staining of the cilia marker (ARL13B) and the SARS-CoV-2 receptor ACE2 (transparent arrowheads). ACE2 staining alone (white arrowheads). Scale bar equals 100 µm. (C) Immunofluorescent staining of formalin fixed paraffin embedded (FFPE) sections of alveolar (ALV) organoids (representative images from patient L2 passage 2), display co-staining of the cilia marker (ARL13B) and the SARS-CoV-2 receptor ACE2 (transparent arrowheads). Scale bar equals 100 µm. ns, not significant.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Human Organotypic Airway and Lung Organoid Cells of Bronchiolar and Alveolar Differentiation Are Permissive to Infection by Influenza and SARS-CoV-2 Respiratory Virus

    doi: 10.3389/fcimb.2022.841447

    Figure Lengend Snippet: Expression of SARS-CoV-2 entry factors in BRON and ALV organoids. (A) ACE2 (left) and TMPRSS2 (right) expression is detected in patient L2 organoids of bronchiolar (BRON) and alveolar (ALV) differentiation from passage 1-4. Relative quantification was performed using the Livac method and alveolar organoids are normalized to the average delta Ct of bronchiolar organoids. The mean value of three technical replicates for each passage are shown as individual data points. GraphPadPrism v9 was used to calculate and display the geometric mean and 90% confidence interval of the biological replicates (passages). Statistics were performed on the delta Ct values using the Mann-Whitney test. No statistically significant differences were observed in the median deltaCt values of ACE2 between BRON and ALV cultures. (B) Immunofluorescent staining of formalin fixed paraffin embedded (FFPE) sections of bronchiolar (BRON) organoids (representative images from patient L2 passage 2), display co-staining of the cilia marker (ARL13B) and the SARS-CoV-2 receptor ACE2 (transparent arrowheads). ACE2 staining alone (white arrowheads). Scale bar equals 100 µm. (C) Immunofluorescent staining of formalin fixed paraffin embedded (FFPE) sections of alveolar (ALV) organoids (representative images from patient L2 passage 2), display co-staining of the cilia marker (ARL13B) and the SARS-CoV-2 receptor ACE2 (transparent arrowheads). Scale bar equals 100 µm. ns, not significant.

    Article Snippet: ACE2 , ACE2 (OTI1G4) #74512 , 1:200 , Cell Signaling.

    Techniques: Expressing, MANN-WHITNEY, Staining, Formalin-fixed Paraffin-Embedded, Marker

    Product information for TaqMan probes used in RT-qPCR assays.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Human Organotypic Airway and Lung Organoid Cells of Bronchiolar and Alveolar Differentiation Are Permissive to Infection by Influenza and SARS-CoV-2 Respiratory Virus

    doi: 10.3389/fcimb.2022.841447

    Figure Lengend Snippet: Product information for TaqMan probes used in RT-qPCR assays.

    Article Snippet: ACE2 , ACE2 (OTI1G4) #74512 , 1:200 , Cell Signaling.

    Techniques: Gene Assay

    The product information and dilution of primary used to detect the various target epitopes in this study.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Human Organotypic Airway and Lung Organoid Cells of Bronchiolar and Alveolar Differentiation Are Permissive to Infection by Influenza and SARS-CoV-2 Respiratory Virus

    doi: 10.3389/fcimb.2022.841447

    Figure Lengend Snippet: The product information and dilution of primary used to detect the various target epitopes in this study.

    Article Snippet: ACE2 , ACE2 (OTI1G4) #74512 , 1:200 , Cell Signaling.

    Techniques: Luciferase

    SDS-PAGE analysis of maltose binding protein-angiotensin converting enzyme 2 N-terminal domain (MBP-ACE2NTD) fusion. (A) A schematic diagram of the fusion protein. (B) Total cellular proteins from IPTG-induced cells (NEB Express) containing the MBP-ACE2NTD fusion. Nine out of 11 clones tested here expressed the fusion (except #1 and #4). Clone #8 was chosen for further study. (C) MBP-ACE2NTD fusion expressed from IPTG induced cells (T7 Shuffle, lanes 1–4). Lane 1, total cellular protein; lane 2, flow-through from an amylose column; lane 3, eluted MBP-ACE2NTD protein from an amylose column; lane 4, MBP-ACE2NTD protein found in the pellet (inclusion body); lane 5, refolded MBP-ACE2NTD fusion protein from cell pellet (NEB Express); M, protein size marker (10–200 kDa, NEB). Arrows indicate the MBP-ACE2NTD protein, a truncated protein, and host MBP. (D) Purified MBP-ACE2NTD protein by amylose magnetic beads. Lane 1, refolded fusion; lanes 2 and 3, protein eluted in a Tris–HCl buffer (20 mM, pH 7.5) and sodium phosphate buffer (0.1 M, pH 8.0) with 10 mM maltose.

    Journal: Frontiers in Microbiology

    Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A

    doi: 10.3389/fmicb.2021.660149

    Figure Lengend Snippet: SDS-PAGE analysis of maltose binding protein-angiotensin converting enzyme 2 N-terminal domain (MBP-ACE2NTD) fusion. (A) A schematic diagram of the fusion protein. (B) Total cellular proteins from IPTG-induced cells (NEB Express) containing the MBP-ACE2NTD fusion. Nine out of 11 clones tested here expressed the fusion (except #1 and #4). Clone #8 was chosen for further study. (C) MBP-ACE2NTD fusion expressed from IPTG induced cells (T7 Shuffle, lanes 1–4). Lane 1, total cellular protein; lane 2, flow-through from an amylose column; lane 3, eluted MBP-ACE2NTD protein from an amylose column; lane 4, MBP-ACE2NTD protein found in the pellet (inclusion body); lane 5, refolded MBP-ACE2NTD fusion protein from cell pellet (NEB Express); M, protein size marker (10–200 kDa, NEB). Arrows indicate the MBP-ACE2NTD protein, a truncated protein, and host MBP. (D) Purified MBP-ACE2NTD protein by amylose magnetic beads. Lane 1, refolded fusion; lanes 2 and 3, protein eluted in a Tris–HCl buffer (20 mM, pH 7.5) and sodium phosphate buffer (0.1 M, pH 8.0) with 10 mM maltose.

    Article Snippet: Mouse monoclonal anti-ACE2 Ab (catalog number #74512) and mouse anti-His tag Ab (catalog number 70796-3) were purchased from Cell Signaling Technologies (CST, MA) and EMC Millipore (Germany), respectively.

    Techniques: SDS Page, Binding Assay, Clone Assay, Marker, Purification, Magnetic Beads

    SDS-PAGE and Western blot analysis of RNase I-ACE2NTD fusion and activity assays. (A) Schematic diagram of RNase I-ACENTD (6×His) fusion. (B) Western blot analysis of RNase I-ACE2NTD in total protein, supernatant (soluble), and refolded protein using anti-His mAb. (C) Same as in (B) , except using anti-ACE2 mAb. (D) SDS-PAGE analysis of the refolded RNase I-ACE2NTD fusion and further purified protein by Ni magnetic beads and Ni spin column. (E) RNase I-ACE2NTD (refolded) ribonuclease activity on fluorescein (FL)-labeled RNA (300 nt) in NEB buffer 3. RNase I (6×His) and MBP-RNase I were used as positive controls. (F) Ribonuclease activity of RNase I-ACE2NTD (purified by Ni magnetic beads or Ni spin column) on SARS-CoV-2 RNA (50 mer). RNase I f , a positive control. FAM-S, FAM-labeled substrate; FAM-P, FAM labeled cleavage product(s).

    Journal: Frontiers in Microbiology

    Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A

    doi: 10.3389/fmicb.2021.660149

    Figure Lengend Snippet: SDS-PAGE and Western blot analysis of RNase I-ACE2NTD fusion and activity assays. (A) Schematic diagram of RNase I-ACENTD (6×His) fusion. (B) Western blot analysis of RNase I-ACE2NTD in total protein, supernatant (soluble), and refolded protein using anti-His mAb. (C) Same as in (B) , except using anti-ACE2 mAb. (D) SDS-PAGE analysis of the refolded RNase I-ACE2NTD fusion and further purified protein by Ni magnetic beads and Ni spin column. (E) RNase I-ACE2NTD (refolded) ribonuclease activity on fluorescein (FL)-labeled RNA (300 nt) in NEB buffer 3. RNase I (6×His) and MBP-RNase I were used as positive controls. (F) Ribonuclease activity of RNase I-ACE2NTD (purified by Ni magnetic beads or Ni spin column) on SARS-CoV-2 RNA (50 mer). RNase I f , a positive control. FAM-S, FAM-labeled substrate; FAM-P, FAM labeled cleavage product(s).

    Article Snippet: Mouse monoclonal anti-ACE2 Ab (catalog number #74512) and mouse anti-His tag Ab (catalog number 70796-3) were purchased from Cell Signaling Technologies (CST, MA) and EMC Millipore (Germany), respectively.

    Techniques: SDS Page, Western Blot, Activity Assay, Purification, Magnetic Beads, Labeling, Positive Control

    Expression of hRNase A-ACE2NTD150 in T7 Express LysY/ lacI q (C3013). (A) Schematic diagram of the fusion protein. (B) SDS-PAGE analysis of five isolates of hRNase A-ACE2NTD150 (five isolates with MBP signal peptide, five clones without MBP signal peptide). Total cell lysate of pET21b serves as a negative control. (C,D) Western blot analysis of the fusion proteins (soluble fraction/supernatant) by anti-His mAb and anti-ACE2 mAb.

    Journal: Frontiers in Microbiology

    Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A

    doi: 10.3389/fmicb.2021.660149

    Figure Lengend Snippet: Expression of hRNase A-ACE2NTD150 in T7 Express LysY/ lacI q (C3013). (A) Schematic diagram of the fusion protein. (B) SDS-PAGE analysis of five isolates of hRNase A-ACE2NTD150 (five isolates with MBP signal peptide, five clones without MBP signal peptide). Total cell lysate of pET21b serves as a negative control. (C,D) Western blot analysis of the fusion proteins (soluble fraction/supernatant) by anti-His mAb and anti-ACE2 mAb.

    Article Snippet: Mouse monoclonal anti-ACE2 Ab (catalog number #74512) and mouse anti-His tag Ab (catalog number 70796-3) were purchased from Cell Signaling Technologies (CST, MA) and EMC Millipore (Germany), respectively.

    Techniques: Expressing, SDS Page, Clone Assay, Negative Control, Western Blot