polyclonal goat anti human ace2 antibody  (R&D Systems)

Journal: Viruses

Article Title: Potential Role of APOBEC3 Family Proteins in SARS-CoV-2 Replication

doi: 10.3390/v16071141

Figure Lengend Snippet: Validation of expression levels for ACE2 protein and A3 mRNAs in THP-1 parent and A3A -to- A3G -null THP-1 cells. ( A ) ACE2 protein expression levels on the surface of THP-1 parent and THP-1#11-4 ( A3A -to- A3G -null THP-1) cells. The ACE2 gene was introduced by a retroviral vector, and the expression levels of the surface ACE2 protein were detected by an anti-ACE2 polyclonal antibody (red). The number in each graph shows the percentage of ACE2 + cells compared to those stained by isotype control (gray). ( B ) A3 mRNA expression levels in Calu-3 (gray), THP-1-ACE2 (blue), and THP-1-ACE2#11-4 (red) cells. A3 mRNA expression levels were quantified by RT-qPCR and normalized to TBP mRNA levels. Each bar represents the average of three independent experiments with Standard deviation (SD). Statistical significance was determined using the two-sided unpaired t -test. *, p < 0.05 compared to THP-1 parent cells.

Article Snippet: A goat anti-ACE2 polyclonal antibody (R&D Systems, Minneapolis, MN, USA, Cat# AF933, 1:50) and an APC-conjugated donkey anti-goat IgG (R&D Systems, Cat# F0108, 1:50) were used for surface ACE2 staining ( A).

Techniques: Biomarker Discovery, Expressing, Retroviral, Plasmid Preparation, Staining, Control, Quantitative RT-PCR, Standard Deviation

Journal: Viruses

Article Title: Potential Role of APOBEC3 Family Proteins in SARS-CoV-2 Replication

doi: 10.3390/v16071141

Figure Lengend Snippet: SARS-CoV-2 replication in THP-1 parent and A3A -to- A3G -null THP-1 cells. ( A ) Replication kinetics of the Wuhan, BA.1, and BA.5 variants produced from THP-1 parent and THP-1#11-4 ( A3A -to- A3G -null THP-1) cells without (blue line) or with (red line) ACE2 protein expression. The SARS-CoV-2 N gene was quantified by RT-qPCR to monitor the viral RNA copy number across the indicated time points. Each timepoint represents the average of four independent experiments with SD. ( B ) Passage experiments. The SARS-CoV-2 N gene in the cell culture supernatants produced from THP-1-ACE2 or THP-1-ACE2#11-4 ( A3A -to- A3G -null THP-1) cells at 96 h postinfection of each passage were quantified by RT-qPCR to monitor the viral RNA copy number of the Wuhan (gray), BA.1 (blue), and BA.5 (red) variants. Each bar represents the average of three independent experiments with SD.

Article Snippet: A goat anti-ACE2 polyclonal antibody (R&D Systems, Minneapolis, MN, USA, Cat# AF933, 1:50) and an APC-conjugated donkey anti-goat IgG (R&D Systems, Cat# F0108, 1:50) were used for surface ACE2 staining ( A).

Techniques: Produced, Expressing, Quantitative RT-PCR, Cell Culture

Journal: Viruses

Article Title: Potential Role of APOBEC3 Family Proteins in SARS-CoV-2 Replication

doi: 10.3390/v16071141

Figure Lengend Snippet: SARS-CoV-2 infectivity produced from THP-1 parent and A3A -to- A3G -null THP-1 cells during passage experiments. ( A ) Representative pictures of plaque assay. Cell culture supernatants obtained from the passage experiments for the Wuhan variant were also used for plaque assay with serial 10-times dilution. ( B ) PFU/mL of the Wuhan variant produced from THP-1-ACE2 (gray) or THP-1-ACE2#11-4 ( A3A -to- A3G -null THP-1) (blue) cells at 96 h postinfection during passage experiments.

Article Snippet: A goat anti-ACE2 polyclonal antibody (R&D Systems, Minneapolis, MN, USA, Cat# AF933, 1:50) and an APC-conjugated donkey anti-goat IgG (R&D Systems, Cat# F0108, 1:50) were used for surface ACE2 staining ( A).

Techniques: Infection, Produced, Plaque Assay, Cell Culture, Variant Assay

Journal: Viruses

Article Title: Potential Role of APOBEC3 Family Proteins in SARS-CoV-2 Replication

doi: 10.3390/v16071141

Figure Lengend Snippet: Analysis of mutations in SARS-CoV-2 genomes produced from THP-1 parent and A3A -to- A3G -null THP-1 cells during passage experiments. SARS-CoV-2 genomic RNA was isolated and subjected to WGS. Sample 1 to 4: Wuhan variant from passage 1 of THP-1-ACE2 cells. Sample 5 to 8: Wuhan variant from passage 5 of THP-1-ACE2 cells. Sample 9 to 12: Wuhan variant from passage 1 of THP-1-ACE2#11-4 ( A3A -to- A3G -null THP-1) cells. Sample 13 and 14: Wuhan variant from passage 3 of THP-1-ACE2#11-4 cells. Green boxes on the top show each SARS-CoV-2 ORF gene with nucleotide position.

Article Snippet: A goat anti-ACE2 polyclonal antibody (R&D Systems, Minneapolis, MN, USA, Cat# AF933, 1:50) and an APC-conjugated donkey anti-goat IgG (R&D Systems, Cat# F0108, 1:50) were used for surface ACE2 staining ( A).

Techniques: Produced, Isolation, Variant Assay

Journal: International Journal of Molecular Sciences

Article Title: Differential Ability of Spike Protein of SARS-CoV-2 Variants to Downregulate ACE2

doi: 10.3390/ijms25021353

Figure Lengend Snippet: Downregulation of ACE2 by the S protein of SARS-CoV-2. ( A ) Cell lysates from ACE2-expressing 293T cells transfected with either an empty vector or an S protein expression vector were subjected to Western blotting using anti-S1, and-ACE2, and anti-β-actin antibodies. Relative band intensities of ACE2 and the S protein are shown in the lower panel. Band intensities of ACE2 or the S protein without the S protein or ACE2 are shown as 1.0, respectively. ( B ) S protein and ACE2-Venus were co-expressed in 293T cells and stained with anti-S1 antibody. ( Left ) ACE2-Venus expression in the absence (red) or presence (blue) of S protein was analyzed in S1-positive cell population. ( Right ) S1 expression in the absence (blue) or presence (yellow) of ACE2-Venus was analyzed. Dotted line represents the no-antibody-control, non-transfected 293T cells.

Article Snippet: Anti-ACE2 goat polyclonal antibody (Cat. AF933) was purchased from R&D Systems (Minneapolis, Minnesota, USA).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Staining, Control

Journal: International Journal of Molecular Sciences

Article Title: Differential Ability of Spike Protein of SARS-CoV-2 Variants to Downregulate ACE2

doi: 10.3390/ijms25021353

Figure Lengend Snippet: Effect of the over-expression of ACE2 in pseudovirus with S protein and authentic SARS-CoV-2 infection. ( A ) Effect of over-expression of ACE2 in pseudovirus infection with SARS-CoV-2 S protein. HIV-1 pseudoviruses with D614G S protein produced in the absence (white bar) or presence (shaded bar) of FLAG-tagged ACE2 were infected into ACE2-expressing 293T cells. The luciferase activities of infected cells were measured after 48 h of infection in triplicate experiments. ( B ) Western blot analyses of pseudovirus-infected cells and the virions in the absence and presence of FLAG-tagged ACE2. Pseudovirus with S protein or VSVG were produced in 293T cells in the absence or presence of ACE2. Cell lysates and pseudoviruses were analyzed by Western blotting using anti-FLAG, anti-S1, anti-VSVG, anti-ACE2, anti-HIV-1 p24, and anti-β-actin antibodies. To monitor the production of psuedovirions, HIV-1 p55 gag precursor and the final product p24 are shown. The positions of the molecular mass marker (kDa) are indicated on the right. ( C ) The 293T cells (No ACE2), ACE2-expressing parental 293T (ACE2 parent), or highly ACE2-expressing 293T cells (ACE2 high) were stained with an anti-ACE2 antibody and analyzed by flow cytometry. ( D ) ACE2 parent and high-ACE2-293T cells were infected with authentic SARS-CoV-2. Viral RNA titers were determined using real-time PCR in the indicated time in triplicate experiments.

Article Snippet: Anti-ACE2 goat polyclonal antibody (Cat. AF933) was purchased from R&D Systems (Minneapolis, Minnesota, USA).

Techniques: Over Expression, Infection, Produced, Expressing, Luciferase, Western Blot, Marker, Staining, Flow Cytometry, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Differential Ability of Spike Protein of SARS-CoV-2 Variants to Downregulate ACE2

doi: 10.3390/ijms25021353

Figure Lengend Snippet: Interaction of ACE2 with the S protein of SARS-CoV-2 in 293T cells. ( A ) Cell lysates from the 293T cells transfected with FLAG-tagged ACE2 and S protein were analyzed by Western blotting using ant-FLAG, anti-S1, anti-β-actin antibodies. Immunoprecipitation (IP) was performed using anti-FLAG and anti-S1 antibodies with protein G-Sepharose beads, and the blots were analyzed with anti-S1 and anti-FLAG antibodies, respectively. The positions of the molecular mass marker (kDa) are indicated on the right. ( B ) Confocal microscopic images of the 293T cells transfected with ACE2-Venus (green) and the S protein. Panels show the counterstaining of cell nuclei with DAPI ( a ), ACE2-Venus ( b ), S protein ( c ), and merged image ( d ). Scale bars correspond to 10 μm.

Article Snippet: Anti-ACE2 goat polyclonal antibody (Cat. AF933) was purchased from R&D Systems (Minneapolis, Minnesota, USA).

Techniques: Transfection, Western Blot, Immunoprecipitation, Marker

Journal: International Journal of Molecular Sciences

Article Title: Differential Ability of Spike Protein of SARS-CoV-2 Variants to Downregulate ACE2

doi: 10.3390/ijms25021353

Figure Lengend Snippet: The degradation of ACE2 by the S protein in the lysosomal compartment via the endocytic pathway. ( A ) ACE2-expressing 293T cells were transfected with the S protein in the presence of various inhibitors targeting protein degradation pathways. Cell lysates were analyzed by Western blotting using anti-S1, anti-ACE2, and anti-β-actin antibodies. The positions of the molecular mass marker (kDa) are indicated on the right. The relative band intensities of ACE2 are shown on the bottom of ACE2 image. The band intensity of ACE2 without the S protein is shown as 1.0. ( B ) Confocal microscopic images of 293T cells transfected with ACE2-Venus and Rab9a-mCherry. The panels show counterstaining with cell nuclei with DAPI ( a ), ACE2-Venus ( b ), Rab9a-mCherry ( c ), and a merged image ( d ) in the absence ( left ) and presence ( right ) of the S protein. Scale bars correspond to 10 μm.

Article Snippet: Anti-ACE2 goat polyclonal antibody (Cat. AF933) was purchased from R&D Systems (Minneapolis, Minnesota, USA).

Techniques: Expressing, Transfection, Western Blot, Marker

Journal: International Journal of Molecular Sciences

Article Title: Differential Ability of Spike Protein of SARS-CoV-2 Variants to Downregulate ACE2

doi: 10.3390/ijms25021353

Figure Lengend Snippet: The differential ability of the S protein from various VOCs to downregulate ACE2. ( A ) ( upper ) Cell lysates from ACE2-expressing 293T cells transfected with S protein from previous VOCs were analyzed by Western blotting using ant-ACE2, anti-S1, and anti-β-actin antibodies. The positions of the molecular mass marker (kDa) are indicated on the right. ( lower ) The relative band intensities of ACE2 in the presence of the S protein from previous VOCs in Western blotting were analyzed and compared using Image Lab software (BioRad) in triplicate experiments. The band intensity of ACE2 without the S protein is shown as 1.0. Bars depicts means and standard deviation (** p < 0.01, *** p < 0.001 using unpaired t -test). ( B ) The S protein and ACE2-Venus were co-expressed in 293T cells and stained with anti-RBD polyclonal antibody. The ACE2 downregulation activity of the S protein of previous VOCs was determined using the mean fluorescence intensity (MFI) of ACE-Venus protein in the S-protein-positive cell population. The column and bar indicate means and standard deviation in triplicate experiments (** p < 0.01 using unpaired t -test). ( C ) S protein and ACE-nLuc were co-expressed in 293T cells in the presence or absence of the S protein from previous VOCs. The ACE2 downregulation activity of the S protein from previous VOCs was calculated using the nanoluciferase activity of the wild-type S proteins as 100% (** p < 0.01, *** p < 0.001 using unpaired t -test).

Article Snippet: Anti-ACE2 goat polyclonal antibody (Cat. AF933) was purchased from R&D Systems (Minneapolis, Minnesota, USA).

Techniques: Expressing, Transfection, Western Blot, Marker, Software, Standard Deviation, Staining, Activity Assay, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Differential Ability of Spike Protein of SARS-CoV-2 Variants to Downregulate ACE2

doi: 10.3390/ijms25021353

Figure Lengend Snippet: ACE2 downregulation and fusion activities of chimeras between the S protein from the Omicron and Delta variants.

Article Snippet: Anti-ACE2 goat polyclonal antibody (Cat. AF933) was purchased from R&D Systems (Minneapolis, Minnesota, USA).

Techniques: Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Differential Ability of Spike Protein of SARS-CoV-2 Variants to Downregulate ACE2

doi: 10.3390/ijms25021353

Figure Lengend Snippet: ACE2 downregulation and fusion activities of Delta, Omicron, and wild-type S with mutations.

Article Snippet: Anti-ACE2 goat polyclonal antibody (Cat. AF933) was purchased from R&D Systems (Minneapolis, Minnesota, USA).

Techniques: Activity Assay