rabbit anti ace2 antibody  (Bioss)

Journal: Frontiers in Microbiology

Article Title: Broad-spectrum inhibition of SARS-CoV-2 variants by dibutyl phthalate through allosteric disruption of Spike-ACE2 interface

doi: 10.3389/fmicb.2025.1610775

Figure Lengend Snippet: Virtual screening of aromatic compounds effectively inhibiting ACE2-Spike interaction. (A) The heatmap of molecular docking structures of 47 aromatic compounds with different con-formations of Spike and ACE2. The color represents the docking binding energy, with a redder color indicating a more stable binding capacity. The top 10 compounds (indicated in red) were utilized for subsequent experimental detections. (B) Structure and classification of 10 potentially effective compounds. (C) The protein pockets (in gray) where the top 10 compounds (in yellow) bind to different conformations of Spike and ACE2. Spike-RBD-1up/Spike-RBD-3down: RBD is highlighted in solid color; Spike-RBD-2up: The S1 and S2 subunits involved in the pocket are emphasized in solid color; RBD-ACE2: RBD is presented in blue, ACE2 in green, the RBM se-quence in direct contact with ACE2 is in pink, and the interface in direct contact with RBD on ACE2 is in red.

Article Snippet: SARS-CoV-2 Spike trimer protein (40589-V08H4), ACE2 protein (10108-H08H), Recombinant Anti-ACE2 Antibody (10108-R003), SARS-CoV-2 (2019-nCoV) Spike Antibody (Rabbit PAb) (40592-T62) were purchased from sino biological (Beijing, China).

Techniques: Binding Assay

Journal: Frontiers in Microbiology

Article Title: Broad-spectrum inhibition of SARS-CoV-2 variants by dibutyl phthalate through allosteric disruption of Spike-ACE2 interface

doi: 10.3389/fmicb.2025.1610775

Figure Lengend Snippet: SPR Analysis of DBP Binding to ACE2 and SARS-CoV-2 Spike Trimer, and Its Inhibition of Spike-ACE2 Interaction. (A,B) The response curves of DBP (0.0122–3.1250 μM) with ACE2 (40 μg/mL, optimized for DBP-ACE2 binding detection) and S trimer (40 μg/mL, optimized for DBP-S trimer binding detection). (C) Concentration-dependent binding of ACE2 (15.625–250 nM) to S trimer (20 μg/mL, optimized for ACE2-S trimer binding detection). (D) Inhibitory effect of DBP on S trimer-ACE2 interaction. K D : Equilibrium dissociation constant. (E) Proposed mechanism of action of DBP.

Article Snippet: SARS-CoV-2 Spike trimer protein (40589-V08H4), ACE2 protein (10108-H08H), Recombinant Anti-ACE2 Antibody (10108-R003), SARS-CoV-2 (2019-nCoV) Spike Antibody (Rabbit PAb) (40592-T62) were purchased from sino biological (Beijing, China).

Techniques: Binding Assay, Inhibition, Concentration Assay

Journal: Frontiers in Microbiology

Article Title: Broad-spectrum inhibition of SARS-CoV-2 variants by dibutyl phthalate through allosteric disruption of Spike-ACE2 interface

doi: 10.3389/fmicb.2025.1610775

Figure Lengend Snippet: Inhibition of SARS-CoV-2 RBD-ACE2 interaction by DBP and its effect on ACE2 enzymatic activity. (A) Schematic illustration of ELISA assays under three experimental conditions: DBP No Premix, DBP-ACE2 Premix, and DBP-Spike Premix. (B) ELISA showing the inhibitory effect of DBP on the binding of SARS-CoV-2 RBD to ACE2. (C) Bar graph depicting the inhibition rate of ACE2/RBD binding by DBP under varied preincubation conditions. DBP at concentrations of 100 μM (blue) and 200 μM (orange) was evaluated in three conditions: no preincubation (DBP No Premix), preincubation with ACE2 (DBP-ACE2 Premix, 1 h), and preincubation with RBD (DBP-RBD Premix, 1 h). (D) Assessment of DBP’s effect on ACE2 enzymatic activity within a concentration range of 12.5–200 μM. Relative Fluorescence units were reported as mean ± SD. Significant differences were observed in the MLN-4760 group compared to the Neg group (*** P < 0.001), while no statistically significant differences (ns) were detected in the other experimental groups. (E) Molecular docking analysis showing that DBP stably binds at the RBD (Cyan). (F) Structural representation of the ACE2-RBD interface (Red) before DBP binding, showing the formation of 18 hydrogen bonds (Yellow) and one salt bridge (Orange). ACE2 inter-action residues are shown in yellow, RBD residues in salmon. (G) Structural representation of the ACE2-RBD interface (Red) after DBP binding, with only 7 hydrogen bonds remaining.

Article Snippet: SARS-CoV-2 Spike trimer protein (40589-V08H4), ACE2 protein (10108-H08H), Recombinant Anti-ACE2 Antibody (10108-R003), SARS-CoV-2 (2019-nCoV) Spike Antibody (Rabbit PAb) (40592-T62) were purchased from sino biological (Beijing, China).

Techniques: Inhibition, Activity Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay, Fluorescence, Stable Transfection

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