rabbit anti ace2  (Sino Biological)

Journal: Nature Communications

Article Title: Presence of SARS-CoV-2 in fetal organs via intraamniotic infection

doi: 10.1038/s41467-025-65131-1

Figure Lengend Snippet: a IF detection of SARS-CoV-2 proteins. b IF staining of human proteins involved in SARS-CoV-2 entry mechanism. Scale bar, 50 μm. n = 2, 2 and 1 specimens for thyroid, thymus and testis, respectively. ACE2 angiotensin-converting enzyme 2, TMPRSS2 transmembrane serine protease 2.

Article Snippet: The following dilutions of primary antibody were used: SARS-CoV-2 nucleocapsid antibody (Cell Signaling Technology, 26369S, USA), 1:1000; SARS-CoV2 orf3a antibody (Novus Biologicals, NBP3-15985, USA), 1:1000; SARS-CoV-2 Spike Glycoprotein S2 antibody (Novus Biologicals, NB100-56578SS), 1:1000; ACE2 antibody (Proteintech, 21115-1-AP), 1:1000; TMPRSS2 antibody (Proteintech, 14437-1-AP), 1:1000; HMGB1 antibody (Proteintech, 10829-1-AP), 1:1000; CD11B antibody (Novus Biologicals, NB110-89474SS), 1:1000; CD86 antibody (Affinity Bioscience, DF6332, China), 1:1000; Fibronectin (FN) antibody (Proteintech, 66042-1-Ig), 1:1000 for IF staining.

Techniques: Staining

Journal: Nature Communications

Article Title: UHRF1 restricts HCoV-229E infection through epigenetic silencing of the viral receptor APN

doi: 10.1038/s41467-025-64977-9

Figure Lengend Snippet: A –D Validation of UHRF1 as a restriction factor across coronavirus genera. Control and UHRF1 -knockout A549-ACE2 cells were infected with alphacoronaviruses (HCoV-NL63, MOI 1, 24 h; SADS-CoV, MOI 1, 24 h) and betacoronavirus (SARS-CoV-2, MOI 0.1, 24 h). UHRF1 -knockout HeLa cells were challenged with betacoronavirus (HCoV-OC43, MOI 1, 24 h), gammacoronavirus (IBV, MOI 1, 24 h), and deltacoronavirus (PDCoV, MOI 0.3, 24 h). Infection efficiency was analyzed by flow cytometry for the percentage of N-positive cells. E –I Proviral effect of UHRF1 on unrelated RNA viruses. UHRF1 -knockout A549 cells were infected with ZIKV (MOI 1, 24 h), SINV (MOI 3, 24 h), VSV (MOI 1, 15 h), H1N1 (MOI 1, 24 h), and EMCV (MOI 0.1, 10 h). Infection efficiency was determined by flow cytometry for the percentage of viral-positive cells. Error bars represent standard deviations from three independent experiments ( n = 3), and each was performed in duplicate. Unpaired, two-sided t-test; mean ± s.d.; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns not significant.

Article Snippet: Control and UHRF1 -knockout A549 cells were detached with TrypLE and incubated with primary antibodies against APN (Invitrogen #14-0138-82, 1 μg/ml), heparan sulfate (10E4) (USBiological #H1890, 1 μg/ml), ACE2 (Sino Biological #10108-RP01, 1:250), DC-SIGN (Biolegend #330102, 1 μg/ml), or TIM-1 (Biolegend #354002, 1 μg/ml) at 4 °C for 25 minutes.

Techniques: Biomarker Discovery, Control, Knock-Out, Infection, Flow Cytometry

Journal: Nature Communications

Article Title: UHRF1 restricts HCoV-229E infection through epigenetic silencing of the viral receptor APN

doi: 10.1038/s41467-025-64977-9

Figure Lengend Snippet: A –C Pseudovirus infection assays. Control and UHRF1 -knockout A549-ACE2 cells were infected with VSV-based pseudoviruses. D –F Virus binding and internalization assays. Cells were incubated with HCoV-229E (MOI 10). Bound or internalized virions were quantified by qRT-PCR or analyzed by confocal microscopy. Representative images from three independent experiments were shown. G , H Western blotting analysis of gene expression in gene-knockout A549 or HeLa cells. Representative images from three independent experiments were shown. I Infection efficiency of HCoV-229E (MOI 0.5, 24 h) in control and UHRF1 -knockout HeLa cells, determined by flow cytometry. J . Relative APN mRNA levels and infection efficiency of HCoV-229E (MOI 1, 12 h) in control and UHRF1 -knockout primary human bronchial epithelial cells (HBEC). mRNA levels were analyzed by qRT-PCR, and infectivity was determined by flow cytometry. K Temporal expression of APN in UHRF1 -knockout A549 cells. Cell lysates were collected at different days post-transduction of sgRNA-expressing lentivirus and analyzed by western blotting. Representative images from three independent experiments were shown. L Surface expression of APN analyzed by flow cytometry in A549 cells edited with control or UHRF1 sgRNA. M Relative APN mRNA levels analyzed by qRT-PCR in A549 cells edited with control or UHRF1 sgRNA. N Relative APN mRNA levels analyzed by qRT-PCR in A549 cells treated with UHRF1 inhibitor UF146 for 2 days. O Control and two APN -knockout A549 clonal cell lines were edited with control or UHRF1 sgRNA, and infected with HCoV-229E (MOI 0.5, 24 h). Infectivity was determined by flow cytometry. P UHRF1 -knockout cells were pre-treated with 5 μg/ml APN-blocking antibody or isotype control for 1 h, then infected with HCoV-229E (MOI 0.5, 24 h) in the presence of antibody. qRT-PCR was performed to determine the relative levels of HCoV-229E N gene. Error bars represent standard deviations from three independent experiments ( n = 3), and each performed in duplicate. Unpaired, two-sided t-test ( A – C , I , J , M ); two-way ANOVA with Sidak’s test ( D, O ); one-way ANOVA with Sidak’s test ( N , P ); mean ± s.d.; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: Control and UHRF1 -knockout A549 cells were detached with TrypLE and incubated with primary antibodies against APN (Invitrogen #14-0138-82, 1 μg/ml), heparan sulfate (10E4) (USBiological #H1890, 1 μg/ml), ACE2 (Sino Biological #10108-RP01, 1:250), DC-SIGN (Biolegend #330102, 1 μg/ml), or TIM-1 (Biolegend #354002, 1 μg/ml) at 4 °C for 25 minutes.

Techniques: Infection, Control, Knock-Out, Virus, Binding Assay, Incubation, Quantitative RT-PCR, Confocal Microscopy, Western Blot, Gene Expression, Gene Knockout, Flow Cytometry, Expressing, Transduction, Blocking Assay

Journal: Nature Communications

Article Title: UHRF1 restricts HCoV-229E infection through epigenetic silencing of the viral receptor APN

doi: 10.1038/s41467-025-64977-9

Figure Lengend Snippet: A UHRF1 does not regulate the expression of exogenous HA-tagged APN and ACE2 from transiently transfected plasmids. Representative Western blotting images from three independent experiments were shown. B Bisulfite sequencing of CpG sites in APN proximal promoter from control and UHRF1 -knockout A549 cells. The methylation (Meth) rate was calculated as the ratio of methylated sites to the total number of sites tested. C A549 cells were treated with 5-AZA for 3 days, and relative APN mRNA levels were determined by qRT-PCR. D , E Huh7 stably expressing DNMT3A were generated by lentivirus transduction. Relative APN mRNA levels were determined by qRT-PCR ( D ), and infectivity was detected by flow cytometry after infection with HCoV-229E (MOI 0.01, 24 h) ( E ). F , G In vitro methylation and dual-luciferase reporter assays. The methylation status of luciferase reporter plasmid was verified by HpaII/MspI digestion ( F ). Representative image from three independent experiments was shown ( F ). The luciferase activity of unmethylated (Unmeth) or methylated (Meth) luciferase reporter plasmid co-transfected with internal control pRL-TK was measured at 24 h post-transfection. Results were normalized to the unmethylated plasmid ( G ). H Electrophoretic mobility shift assay (EMSA). Nuclear extracts were incubated with biotin-labeled unmethylated or methylated APN promoter probe to detect DNA-protein complexes. Representative images from three independent experiments were shown. I , J Chromatin immunoprecipitation (ChIP) assay with c-Maf expression. qPCR was performed to detect c-Maf binding to the transcription factor (TF) binding site ( I ) or CpG island ( J ) of the APN proximal promoter. K Schematic diagram of UHRF1 truncations. L , M UHRF1 -knockout A549 stably expressing wild-type or truncated UHRF1 were established by lentivirus transduction and verified by western blotting ( L ). Representative images from three independent experiments were shown ( L ). Cells were infected with HCoV-229E (MOI 0.5, 24 h) at day 10 post-transduction, and the infectivity was determined by flow cytometry ( M ). Error bars represent standard deviations from three independent experiments ( n = 3), and each performed in duplicate. One-way ANOVA with Sidak’s test ( C , M ); unpaired, two-sided t-test ( D , E , G , I – J ); mean ± s.d.; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: Control and UHRF1 -knockout A549 cells were detached with TrypLE and incubated with primary antibodies against APN (Invitrogen #14-0138-82, 1 μg/ml), heparan sulfate (10E4) (USBiological #H1890, 1 μg/ml), ACE2 (Sino Biological #10108-RP01, 1:250), DC-SIGN (Biolegend #330102, 1 μg/ml), or TIM-1 (Biolegend #354002, 1 μg/ml) at 4 °C for 25 minutes.

Techniques: Expressing, Transfection, Western Blot, Methylation Sequencing, Control, Knock-Out, Methylation, Quantitative RT-PCR, Stable Transfection, Generated, Transduction, Infection, Flow Cytometry, In Vitro, Luciferase, Plasmid Preparation, Activity Assay, Electrophoretic Mobility Shift Assay, Incubation, Labeling, Chromatin Immunoprecipitation, Binding Assay

Journal: Nature Communications

Article Title: UHRF1 restricts HCoV-229E infection through epigenetic silencing of the viral receptor APN

doi: 10.1038/s41467-025-64977-9

Figure Lengend Snippet: A Volcano plot of RNA-seq analysis. Total cellular RNA was extracted from control and UHRF1 -knockout A549-ACE2 cells and subjected to RNA-seq. Genes with an absolute Log 2 fold change >2 and adjusted P -value < 0.05 were considered as differentially expressed. Differential expression analysis was performed using DESeq2 with a two-sided Wald test. P -values were adjusted for multiple comparisons using the Benjamini-Hochberg method. B Schematic of focused CRISPR activation screening. A sub-library targeting 2172 of the 2210 upregulated genes identified from RNA-seq analysis of UHRF1 -knockout cells, with ~4 sgRNAs per gene, was generated and transduced into A549-ACE2-dCas9 cells. Cells were infected with HCoV-229E-mGreen (MOI 0.5, 24 h) or SARS-CoV-2 transcription- and replication-competent virus-like particles in which the N gene is replaced by the reporter GFP (trVLP-GFP) (MOI 0.5, 24 h). Infected reporter-positive cells were sorted for genomic DNA extraction and sgRNA sequence analysis. Created in BioRender. Wang, P. (2025) https://BioRender.com/35yt09k . C , D Genes identified from CRISPR screens for HCoV-229E ( C ) and SARS-CoV-2 ( D ). Genes were analyzed by MAGeCK software and sorted based on -log 10 (MAGeCK score) and P -values. The algorithm employs a one-sided test to identify genes under positive selection, and P -values were adjusted for multiple testing using the Benjamini-Hochberg method.

Article Snippet: Control and UHRF1 -knockout A549 cells were detached with TrypLE and incubated with primary antibodies against APN (Invitrogen #14-0138-82, 1 μg/ml), heparan sulfate (10E4) (USBiological #H1890, 1 μg/ml), ACE2 (Sino Biological #10108-RP01, 1:250), DC-SIGN (Biolegend #330102, 1 μg/ml), or TIM-1 (Biolegend #354002, 1 μg/ml) at 4 °C for 25 minutes.

Techniques: RNA Sequencing, Control, Knock-Out, Quantitative Proteomics, CRISPR, Activation Assay, Generated, Infection, Virus, DNA Extraction, Sequencing, Software, Selection

Journal: Journal of Advanced Research

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

doi: 10.1016/j.jare.2024.12.010

Figure Lengend Snippet: Glycan profiles of the SARS-CoV-2 S1 and the ACE2 receptor. (A) Schematic diagram illustrating the process of preparing antibody-overlay lectin microarrays. (B, C) Scanned images were obtained for the analysis of glycopatterns from the SARS-CoV-2-S1 (B) and ACE2 (C). HEK293-expressing recombinant proteins of S1 and ACE2 were incubated with lectin microarrays. Subsequently, the microarrays were incubated with biotin-labeled primary antibodies and Cy3-labeled streptavidin. The representative lectins that recognized β1-4 galactosylated glycans (ECA and MAL-I), agalactosylated glycans (GSL-II and STL), bisected and bi-antennary N-glycans (PHA-E), oligo-mannose type N-glycans (ConA and HHL), fucosylation (PSA and LCA) and α-2,3 linked sialic acid (MAL-II) were marked with white frames. (D, E) Analysis of glycopatterns on S1 (D) and ACE2 (E). The lectins were classified according to their glycan binding preferences. The NFIs of each lectin were obtained from three biological replicates. The proportion of main types of glycans was calculated by diverging the sum of the NFIs of the lectins that recognized this type of glycan by the total NFIs of all lectins. Blue square: GlcNAc; yellow circle: galactose; yellow square: GalNAc; green circle: mannose; red triangle: fucose; purple diamond: sialic acid. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The primary antibodies used were as follows: a mouse monoclonal antibody against ACE2 (Proteintech, China), a rabbit polyclonal antibody against the SARS-CoV-2 S protein (ABclonal, China), and a mouse monoclonal antibody against GAPDH (Abways, China).

Techniques: Glycoproteomics, Expressing, Recombinant, Incubation, Labeling, Binding Assay

Journal: Journal of Advanced Research

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

doi: 10.1016/j.jare.2024.12.010

Figure Lengend Snippet: Role of N-glycans in the interaction between S1 and ACE2. (A) Schematic diagram illustrating the process of manufacturing the SRAS-CoV-2-related recombinant protein microarrays. (B, C) The N-glycans on S1 of SARS-CoV-2/1 and ACE2 were removed by PNGase F glycosidase. The roles of N-glycans in the interaction between the SARS-CoV-2-S1 /ACE2 (B) and the SARS-CoV-1-S1/ACE2 (C) were evaluated using protein microarrays. Statistical analysis of the relative fluorescence intensities was conducted by comparing the PNGase F-treated S1 and ACE2 to the intact glycosylated protein using one-way ANOVA with Dunnett multiple comparisons. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated. (D) MD simulation of the interaction between the trimeric S protein and ACE2. The distances between the N-glycosites and the center of the binding interface (represented by the green globule) within 50 Å were marked with red spheres. Other N-glycosites were marked with yellow spheres. (E) The interactions of glycans at specific sites and GRDs (marked with a red frame) may be involved in the binding of the S protein to ACE2. (F) MD simulated the interactions of glycans at specific sites and GRDs. The distances between the terminal glycans on these sites and the three GRDs on the ACE and S1 subunit were monitored during a 100 ns MD simulation. The distances of N546-GRD1, N322-GRD2, and N53-GRD2 fluctuated between 1 and 15 Å, while the distances of N343-GRD3 and N165-GRD3 fluctuated between 20 and 35 Å. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The primary antibodies used were as follows: a mouse monoclonal antibody against ACE2 (Proteintech, China), a rabbit polyclonal antibody against the SARS-CoV-2 S protein (ABclonal, China), and a mouse monoclonal antibody against GAPDH (Abways, China).

Techniques: Recombinant, Fluorescence, Binding Assay

Journal: Journal of Advanced Research

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

doi: 10.1016/j.jare.2024.12.010

Figure Lengend Snippet: β1-4 galactosylated N-glycans of ACE2 mediated the binding of S1 of SARS-CoV-2 and its variants. (A) Molecular docking analysis of S1 and ACE2 with various saccharides. The potential binding capacities of S1 of SARS-CoV-2 (Wuhan-Hu-1 strain, wild type) and its variants (Delta and Omicron), as well as ACE2, to various saccharides were predicted by molecular docking analysis. The saccharides were listed in columns, S1 and ACE2 were listed in rows. The different binding abilities were represented by the values of binding free energy, which were indicated by the color of each square: red: high affinity, blue: low affinity, Xyl: xylose, Glc: glucose; Man: mannose; GlcNAc: N-acetylglucosamine, GalNAc: N-acetylgalactosamine; SA: sialic acid. (B) Validation of β1-4 galactosylation level in intact and de-β1-4galactosylated ACE2. After β1-4 galactosidase treatment, the level of β1-4 galactosylation on ACE2 was detected by lectin blotting of MAL-I. The protein level of ACE2 served as the control. (C) Scanning images of protein microarrays incubated with 1 μg of intact or de-β1-4galactosylated ACE2. (D) Effect of β1-4 galactosylation of ACE2 on the binding of S1 to ACE2. The relative fluorescence intensities were statistically analyzed by comparing the de-β1-4galactosylated ACE2 to intact ACE2 using an unpaired t test with Welch's correction. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The primary antibodies used were as follows: a mouse monoclonal antibody against ACE2 (Proteintech, China), a rabbit polyclonal antibody against the SARS-CoV-2 S protein (ABclonal, China), and a mouse monoclonal antibody against GAPDH (Abways, China).

Techniques: Binding Assay, Biomarker Discovery, Control, Incubation, Fluorescence

Journal: Journal of Advanced Research

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

doi: 10.1016/j.jare.2024.12.010

Figure Lengend Snippet: Evaluation of the ability of free saccharides to block S1 and ACE2 binding. (A, B) Scanning images of protein microarrays. ACE2 was mixed with GalNAc (A) or Galβ1-3GalNAc (B), and the inhibitory effect of saccharides was evaluated using protein microarrays. (C, D) Effect of GalNAc (C) and Galβ-1,3GalNAc (D) on the interaction between S1 of SARS-CoV-2/1 and ACE2. The binding signals were extracted, and the relative fluorescence intensities were compared with those of the controls using one-way ANOVA with Dunnett multiple comparisons. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated.

Article Snippet: The primary antibodies used were as follows: a mouse monoclonal antibody against ACE2 (Proteintech, China), a rabbit polyclonal antibody against the SARS-CoV-2 S protein (ABclonal, China), and a mouse monoclonal antibody against GAPDH (Abways, China).

Techniques: Blocking Assay, Binding Assay, Fluorescence

Journal: Journal of Advanced Research

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

doi: 10.1016/j.jare.2024.12.010

Figure Lengend Snippet: Evaluation of isolated glycoproteins for the inhibition of S1 and ACE2 binding. (A) The scanned image was obtained from the lectin microarray analysis of glycoproteins isolated from bovine milk. The representative lectins that recognized β1-4 galactosylated glycans (ECA and MAL-I), agalactosylated glycans (GSL-II), bisected N-glycans (PHA-E), high-mannose glycans (ConA), fucosylation (AAL, PSA, and LCA), α2-3 linked sialic acid (MAL-II), and α2-6 linked sialic acid (SNA) were marked with white frames. (B) Analysis of glycopatterns on isolated glycoproteins. The lectins were classified according to their glycan binding preferences. The NFIs of each lectin were obtained from three biological replicates. The proportion of galactosylated glycans was calculated by diverging the sum of the NFIs of the lectins that recognized Gal/GalNAc by the total NFIs. (C, D) Evaluation of the effect of intact and de-sialylated isolated glycoproteins on the interaction between S1 of SARS-CoV-2/1 and ACE2. The intact isolated glycoproteins (C) or de-sialylated isolated glycoproteins (D) were mixed with ACE2 and incubated with protein microarrays. The relative binding intensities of each group were compared with those of the control group, and any significant differences between groups were determined using one-way ANOVA with Dunnett multiple comparisons. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated. (E) Inhibition curves for intact isolated glycoproteins (upper) and de-sialylated isolated glycoproteins (lower). Four-parameter inhibition curves were generated, and the particular IC50 values for intact isolated glycoproteins and de-sialylated isolated glycoproteins were indicated in this graph. The data were obtained from three biological replicates and presented as the mean ± SD (error bars).

Article Snippet: The primary antibodies used were as follows: a mouse monoclonal antibody against ACE2 (Proteintech, China), a rabbit polyclonal antibody against the SARS-CoV-2 S protein (ABclonal, China), and a mouse monoclonal antibody against GAPDH (Abways, China).

Techniques: Isolation, Inhibition, Binding Assay, Microarray, Glycoproteomics, Incubation, Control, Generated