ace2 goat anti hace2  (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: 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: Viruses

Article Title: Stabilization of the Metastable Pre-Fusion Conformation of the SARS-CoV-2 Spike Glycoprotein through N-Linked Glycosylation of the S2 Subunit

doi: 10.3390/v16020223

Figure Lengend Snippet: α-glycosidase inhibitors impaired SARS-CoV-2 S-mediated membrane fusion. ( A ) HEK293T cells transfected with plasmids expressing the SARS-CoV-2 S protein were treated with 100 µg/mL castanospermine or NB-DNJ for 40 h, after which the expression levels of S protein in cell lysates were detected using Western blot analysis with antibodies against the S2 subunit of the SARS-CoV-2 S protein. β-actin was used as a loading control. ( B ) HEK293T cells expressing the SARS-CoV-2 S protein and GFP were treated with α-glycosidase inhibitors for 40 h and then trypsinized at 40 h post-transfection and co-cultured with HEK293/hACE2 for another 4 h, then imaged using a fluorescence microscope. The area of syncytia in each image was measured using ImageJ. An unpaired two-tailed Student’s t -test was used for statistical analysis, ** p < 0.01; * p < 0.05. ( C ) Effect of α-glycosidase inhibitors on the proliferation of HEK293T cells. HEK293T cells were treated with 100 µg/mL castanospermine or NB-DNJ for 40 h, after which cell viability was determined using the CCK8 assay. ( D ) Western blot analysis of SARS-CoV-2 S-pseudotyped lentiviral particles produced in HEK293T cells in the absence or presence of α-glycosidase inhibitors. p24 was used as a loading control. ( E ) The transduction efficiency of SARS-CoV-2 S-pseudotyped lentiviral particles produced in HEK293T cells in the absence or presence of α-glycosidase inhibitors. An unpaired two-tailed Student’s t -test was used for statistical analysis, ** p < 0.01.

Article Snippet: Subsequently, the cells were washed and incubated with 1 µg/mL polyclonal goat anti-human ACE2 antibody (R&D Systems, Minneapolis, MN, USA) for 1 h. Next, the cells were incubated with FITC-conjugated rabbit anti-goat secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. After washing, the cells were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Membrane, Transfection, Expressing, Western Blot, Cell Culture, Fluorescence, Microscopy, Two Tailed Test, CCK-8 Assay, Produced, Transduction

Journal: Viruses

Article Title: Stabilization of the Metastable Pre-Fusion Conformation of the SARS-CoV-2 Spike Glycoprotein through N-Linked Glycosylation of the S2 Subunit

doi: 10.3390/v16020223

Figure Lengend Snippet: The expression levels and receptor-binding capacity of the N-glycosylation-site mutants of the SARS-CoV-2 S protein. ( A ) Schematic representation of N-linked glycosylation sites in the SARS-CoV-2 S protein. ( B ) HEK293T cells were transfected with plasmids encoding wild-type S protein or with N-glycosylation-site mutants. After 40 h of transfection, the cells were lysed and analyzed via Western blot with antibodies against the SARS-CoV-2 S2 subunit. β-actin was used as a loading control. ( C ) Cell-surface expression of wild-type S protein and different N-glycosylation-site mutants, determined via flow cytometry. ( D ) HEK293T cells were transfected with plasmids encoding wild-type S protein or different N-glycosylation mutants. After 40 h of transfection, the cells were detached with 1 mM EDTA, incubated with soluble hACE2 for 1 h on ice, incubated with primary antibodies against hACE2, and then incubated with FITC-conjugated secondary antibodies. The cells were then subjected to flow cytometry.

Article Snippet: Subsequently, the cells were washed and incubated with 1 µg/mL polyclonal goat anti-human ACE2 antibody (R&D Systems, Minneapolis, MN, USA) for 1 h. Next, the cells were incubated with FITC-conjugated rabbit anti-goat secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. After washing, the cells were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Expressing, Binding Assay, Transfection, Western Blot, Flow Cytometry, Incubation

Journal: Viruses

Article Title: Stabilization of the Metastable Pre-Fusion Conformation of the SARS-CoV-2 Spike Glycoprotein through N-Linked Glycosylation of the S2 Subunit

doi: 10.3390/v16020223

Figure Lengend Snippet: Cell–cell fusion capacity of N-glycosylation mutants of the SARS-CoV-2 S protein. ( A ) HEK293T cells were co-transfected with plasmids encoding either wild-type S protein or different N-glycosylation-site-mutant S proteins and plasmids encoding GFP. After 40 h of transfection, the cells were trypsinized and co-cultured with HEK293/hACE2 cells for an additional 4 h and then imaged using a fluorescence microscope. ( B ) Quantification of cell–cell fusion. The total number of nuclei and the number of nuclei in fused cells for each image were counted. The fusion efficiency was calculated as the number of nuclei in syncytia/the total number of nuclei ×100.

Article Snippet: Subsequently, the cells were washed and incubated with 1 µg/mL polyclonal goat anti-human ACE2 antibody (R&D Systems, Minneapolis, MN, USA) for 1 h. Next, the cells were incubated with FITC-conjugated rabbit anti-goat secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. After washing, the cells were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Transfection, Mutagenesis, Cell Culture, Fluorescence, Microscopy

Journal: Viruses

Article Title: Stabilization of the Metastable Pre-Fusion Conformation of the SARS-CoV-2 Spike Glycoprotein through N-Linked Glycosylation of the S2 Subunit

doi: 10.3390/v16020223

Figure Lengend Snippet: Removal of N-glycans from the SARS-CoV-2 S protein destabilized the S protein. ( A ) HEK293T cells were co-transfected with plasmids encoding EGFP and plasmids encoding wild-type SARS-CoV-2 S protein, the N1074Q mutant, or the T1076A mutant. After 40 h of transfection, the cells were visualized using a fluorescence microscope. ( B ) Endogenous ACE2 knockout in HEK293T cells was confirmed through DNA sequencing. A single-nucleotide insertion in the ACE2 gene caused a frameshift and led to the premature termination of ACE2 translation. ( C ) The endogenous-ACE2-knockout HEK293T cells were co-transfected with plasmids encoding EGFP and plasmids encoding wild-type SARS-CoV-2 S protein, the N1074Q mutant, or the T1076A mutant. After 40 h of transfection, the cells were visualized using a fluorescence microscope. ( D , E ) HEK293T cells were transfected with plasmids encoding wild-type SARS-CoV-2 S protein, the N1074Q mutant, or the T1076A mutant. After 40 h of transfection, the S protein in cell lysates ( D ) and supernatants ( E ) was analyzed via Western blot with antibodies against the S2 subunit and the RBD subunit of the SARS-CoV-2 S protein, respectively.

Article Snippet: Subsequently, the cells were washed and incubated with 1 µg/mL polyclonal goat anti-human ACE2 antibody (R&D Systems, Minneapolis, MN, USA) for 1 h. Next, the cells were incubated with FITC-conjugated rabbit anti-goat secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. After washing, the cells were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Transfection, Mutagenesis, Fluorescence, Microscopy, Knock-Out, DNA Sequencing, Western Blot

Journal: Viruses

Article Title: Stabilization of the Metastable Pre-Fusion Conformation of the SARS-CoV-2 Spike Glycoprotein through N-Linked Glycosylation of the S2 Subunit

doi: 10.3390/v16020223

Figure Lengend Snippet: Removal of the furin cleavage site compensated for the instability of the N-glycosylation mutant N1074Q. ( A ) HEK293T cells were transfected with plasmids encoding wild-type SARS-CoV-2 S protein, wild-type S protein with a mutant furin cleavage site (WT-delFurin), mutant N1074Q, or N1074Q with a mutant furin cleavage site (N1074Q-delFurin). After 40 h of transfection, the cells were lysed and analyzed via Western blot with antibodies against the SARS-CoV-2 S2 subunit. β-actin was used as a loading control. ( B ) HEK293T cells were co-transfected with plasmids encoding GFP and plasmids encoding wild-type SARS-CoV-2 S protein, the WT-delFurin mutant, the N1074Q mutant, or the N1074Q-delFurin mutant. After 40 h of transfection, the cells were visualized using a fluorescence microscope. ( C ) A cell–cell fusion assay was performed to assess the fusogenicity of wild-type SARS-CoV-2 S protein, the WT-delFurin mutant, the N1074Q mutant, and the N1074Q-delFurin mutant. HEK293T cells were co-transfected with plasmids encoding wild-type or mutant S proteins and plasmids encoding GFP. After 30 h of transfection, the cells were trypsinized and co-cultured with HEK293/hACE2 cells for an additional 4 h and then imaged using a fluorescence microscope.

Article Snippet: Subsequently, the cells were washed and incubated with 1 µg/mL polyclonal goat anti-human ACE2 antibody (R&D Systems, Minneapolis, MN, USA) for 1 h. Next, the cells were incubated with FITC-conjugated rabbit anti-goat secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. After washing, the cells were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Mutagenesis, Transfection, Western Blot, Fluorescence, Microscopy, Cell-Cell Fusion Assay, Cell Culture

Journal: Viruses

Article Title: Stabilization of the Metastable Pre-Fusion Conformation of the SARS-CoV-2 Spike Glycoprotein through N-Linked Glycosylation of the S2 Subunit

doi: 10.3390/v16020223

Figure Lengend Snippet: Removal of N-glycans from the S2 subunit of SARS-CoV S protein destabilized the S protein. ( A ) Amino-acid sequence alignment of S2 subunits from SARS-CoV and SARS-CoV-2. Red asterisks indicate N-linked glycosylation sites. ( B ) HEK293T cells were transfected with an empty vector or with plasmids encoding the wild-type SARS-CoV S protein or one of the various glycosylation mutants. After 40 h of transfection, the cells were lysed and analyzed with polyclonal rabbit anti-SARS S1 antibodies T62. β-actin was used as a loading control. ( C ) HEK293T cells were co-transfected with plasmids encoding EGFP and plasmids encoding wild-type SARS-CoV S protein or one of the various glycosylation mutants. After 40 h of transfection, the cells were visualized using a fluorescence microscope. ( D ) HEK293T/ACE2-KO cells were co-transfected with plasmids encoding EGFP and plasmids encoding wild-type SARS-CoV S protein, the N699Q mutant, or the N1080Q mutant. After 40 h of transfection, the cells were visualized using a fluorescence microscope.

Article Snippet: Subsequently, the cells were washed and incubated with 1 µg/mL polyclonal goat anti-human ACE2 antibody (R&D Systems, Minneapolis, MN, USA) for 1 h. Next, the cells were incubated with FITC-conjugated rabbit anti-goat secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. After washing, the cells were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Techniques: Sequencing, Transfection, Plasmid Preparation, Fluorescence, Microscopy, Mutagenesis