rabbit polyclonal antibody (pab) anti-ace2  (Abcan Audio Visual Inc)

Journal: Frontiers in Immunology

Article Title: Simian Immunodeficiency Virus Infection Mediated Changes in Jejunum and Peripheral SARS-CoV-2 Receptor ACE2 and Associated Proteins or Genes in Rhesus Macaques

doi: 10.3389/fimmu.2022.835686

Figure Lengend Snippet: mRNA expression of ACE2 associated proteins and inflammatory cytokines during pre and post infection. mRNA expression of protein of interest ( ACE2 , TMPRSS2 , AGTR1 , AGTR2 , and ADAM17 ) and inflammatory cytokines ( IL-1β , IL-6 , and TNF-α) in whole jejunum tissue were quantified. mRNA expression for each gene was quantified relative to the internal control GAPDH (n=6-8). The error bars represent the mean of relative fold-change for each group ± SE. * p < 0.05, ** p < 0.01 as determined by the unpaired T-test. The green, blue, and red bar graphs represent pre, acute and chronic infection time points, respectively.

Article Snippet: Tissue sections of 5 μM thickness were stained by incubating for 1h with rabbit polyclonal anti-ACE2 antibodies (Sino Biologicals, USA), then washed and stained for 40 min with Alexa Flour 568-conjugated secondary antibodies (Life Technologies, USA) ( ).

Techniques: Expressing, Infection

Journal: Frontiers in Immunology

Article Title: Simian Immunodeficiency Virus Infection Mediated Changes in Jejunum and Peripheral SARS-CoV-2 Receptor ACE2 and Associated Proteins or Genes in Rhesus Macaques

doi: 10.3389/fimmu.2022.835686

Figure Lengend Snippet: IL-6 mRNA expression negatively correlated with jejunum ACE2 expression during infection. mRNA expression of ACE2 and IL- 6 in the jejunum were measured by qRT-PCR, and correlations between these two genes at different infection stages were analyzed. (A) Spearman’s rank correlation analysis between ACE and IL- 6 during pre (green circles) and acute (blue circles) infection showed a significant negative correlation (r = -0.685, p = 0.017) with the increase in IL-6 and the reduction of ACE2 expression (n=6). (B) Similarly, a significant negative correlation was detected between IL-6 and ACE2 mRNA expression during pre infection (green circles) and chronic infection (red circles) (r= -0.720, p = 0.007) (n=6-8).

Article Snippet: Tissue sections of 5 μM thickness were stained by incubating for 1h with rabbit polyclonal anti-ACE2 antibodies (Sino Biologicals, USA), then washed and stained for 40 min with Alexa Flour 568-conjugated secondary antibodies (Life Technologies, USA) ( ).

Techniques: Expressing, Infection, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Simian Immunodeficiency Virus Infection Mediated Changes in Jejunum and Peripheral SARS-CoV-2 Receptor ACE2 and Associated Proteins or Genes in Rhesus Macaques

doi: 10.3389/fimmu.2022.835686

Figure Lengend Snippet: Global transcriptomic profiling of enteroids from infected and uninfected RMs. Enteroids were grown after isolating crypts from the jejunum and total RNA was isolated for the RNA-seq analysis. (A) PCA plot of enteroids from 5 uninfected (JK56, KA42, KA76, KA78, and KP54) and 5 chronically infected RMs (KA42, KA78, KP54, KH79, and KM05) revealed a clear separation between infected RMs and uninfected controls, along with the first principal component (PC1) with 43% of the total variance and the second principal component (PC2) with 25%. (B) Heatmap of differentially expressed genes (DEGs) from enteroids which encode RAS-related proteins, arranged from smallest to largest adjusted p-values. Note that there were no significant differences in TMPRSS2 , AGTR1 , AGTR2 , and AGT gene expression between infected and uninfected enteroids. P- and C-suffix of animal numbers at the bottom of Heatmap denote pre and chronic infection time points, respectively. Spearman’s rank correlation coefficient of determination between ACE2 and DPP4 gene expression (C) , ACE2 and MME gene expression (D) , ACE2 and ANPEP gene expression (E) , and ACE2 and ENPEP gene expression (F) is shown for all 5 uninfected and 5 infected macaques. Strong significantly positive correlations were detected between decreased ACE gene expression and reduction of DPP4 , ANPEP , or ENPEP expression in enteroids. A positive correlation was also detected between ACE2 and MME gene expression, but it was not statistically significant. (C-F) Green and red circles represent pre and chronic infection timepoints, respectively.

Article Snippet: Tissue sections of 5 μM thickness were stained by incubating for 1h with rabbit polyclonal anti-ACE2 antibodies (Sino Biologicals, USA), then washed and stained for 40 min with Alexa Flour 568-conjugated secondary antibodies (Life Technologies, USA) ( ).

Techniques: Infection, Isolation, RNA Sequencing Assay, Expressing

Journal: Frontiers in Immunology

Article Title: Simian Immunodeficiency Virus Infection Mediated Changes in Jejunum and Peripheral SARS-CoV-2 Receptor ACE2 and Associated Proteins or Genes in Rhesus Macaques

doi: 10.3389/fimmu.2022.835686

Figure Lengend Snippet: Negative correlation between the expression of SOX9 and other important DEGs in the enteroid. The two-tailed Spearman’s correlation coefficient analyses between the expression of SOX9 and ACE2 (A) , SOX9 and DPP4 (B) , SOX9 and ANPEP (C) , and SOX9 and ENPEP (D) were performed using read counts obtained after transcriptomic analysis of the enteroids at pre (0 dpi) and chronic (180 dpi) infection for 5 subjects. SOX 9 expression was upregulated in SIV infection compared to pre infection. The correlation and significant values are shown for each plot. Green and red open circles represent pre (0 dpi) and chronic (180 dpi) infection time points, respectively. P value < 0.05 is considered statistically significant. Significant negative correlation was detected between SOX9 and other important DEGs.

Article Snippet: Tissue sections of 5 μM thickness were stained by incubating for 1h with rabbit polyclonal anti-ACE2 antibodies (Sino Biologicals, USA), then washed and stained for 40 min with Alexa Flour 568-conjugated secondary antibodies (Life Technologies, USA) ( ).

Techniques: Expressing, Two Tailed Test, Infection

Journal: Frontiers in Immunology

Article Title: Simian Immunodeficiency Virus Infection Mediated Changes in Jejunum and Peripheral SARS-CoV-2 Receptor ACE2 and Associated Proteins or Genes in Rhesus Macaques

doi: 10.3389/fimmu.2022.835686

Figure Lengend Snippet: Upstream regulator analysis predicts key regulators for gene expression. (A) Upstream regulators for the gene ACE2 as analyzed by IPA. A total of 26 predicted upstream regulators were identified as possible contributors to the change in ACE2 mRNA expression after infection. Known links between ACE2 and the predicted upstream regulators are indicated. Different colors indicate the predicted relationships between the regulators and ACE2 gene expression. (B) HNF1A was found to be a common upstream inhibitory regulator for ACE2 , DPP4 , ENPEP , and ANPEP gene expression.

Article Snippet: Tissue sections of 5 μM thickness were stained by incubating for 1h with rabbit polyclonal anti-ACE2 antibodies (Sino Biologicals, USA), then washed and stained for 40 min with Alexa Flour 568-conjugated secondary antibodies (Life Technologies, USA) ( ).

Techniques: Expressing, Infection

Journal: Frontiers in Immunology

Article Title: Simian Immunodeficiency Virus Infection Mediated Changes in Jejunum and Peripheral SARS-CoV-2 Receptor ACE2 and Associated Proteins or Genes in Rhesus Macaques

doi: 10.3389/fimmu.2022.835686

Figure Lengend Snippet: No significant changes in ACE2 protein expression detected in jejunum during SIV infection. Representative isotype control for ACE2 showing the absence of nonspecific background staining ( A , KM05). Representative immunofluorescence images of ACE2 expression detected in RM KM05 during pre (B) , acute (21 dpi, C ), and chronic (180 dpi, D ) infection. Expressions of ACE2 proteins in the jejunum epithelium are shown by yellow arrows. (E) Co-localization of ACE2 (green) and cytokeratin (red), indicated by orange arrow at the epithelial barrier, showed ACE2 expression only at the intestinal brush border. DAPI stains the cell nucleus. (F) Scatter plots (indicating mean ± SE) of ACE2 immunofluorescence pixel values for pre, acute (21 dpi), and chronic (180 dpi) infection (n=6). An average of 20-23 regions of interest (20X objective) was randomly selected from villi from each animal to quantify ACE2 expression, and the mean intensity of these regions for each individual is represented by each point on the scatter plot. Each animal represents a different shape.

Article Snippet: Tissue sections of 5 μM thickness were stained by incubating for 1h with rabbit polyclonal anti-ACE2 antibodies (Sino Biologicals, USA), then washed and stained for 40 min with Alexa Flour 568-conjugated secondary antibodies (Life Technologies, USA) ( ).

Techniques: Expressing, Infection, Staining, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Simian Immunodeficiency Virus Infection Mediated Changes in Jejunum and Peripheral SARS-CoV-2 Receptor ACE2 and Associated Proteins or Genes in Rhesus Macaques

doi: 10.3389/fimmu.2022.835686

Figure Lengend Snippet: Loss of jejunum lamina propria CD4+ T-cells correlates with increased AGTR2 and decreased TMPRSS2 expression during infection. (A) Representative contour plots showing loss of jejunum lamina propria CD4+ T cells during acute (21 dpi) and chronic (180 dpi) compared to pre (0 dpi) infection time point. In each plot, the percentage of CD4 and CD8+ T cells are shown in the top left and lower right position, respectively. (B) Scatter plots showing percentages of CD4+ T cells (mean ± SE) from jejunum LPL with significant loss of CD4+ T cells during acute and chronic infection compared to pre time points (n=10). Each animal is symbolized by a different shape. Asterisks indicate statistical differences between stages of infection as calculated by Bonferroni analysis (*** p < 0.001 and **** p < 0.0001). A two-tailed Spearman’s correlation coefficient analysis between percentages of CD4+ and ACE2 mean fluorescence intensity (C) , percentage of CD4+ and AGTR2+ cells (D) , CD4+ and TMPRSS2+ in villi (E) , and CD4+ and TMPRSS2+ in crypts (F) is shown for 6 individuals at pre, acute, and chronic infection. Green, blue, and red open circles denote pre, acute, and chronic infection time points, respectively. Significant negative and positive correlation was detected between percentages of CD4+ T cells and AGTR2+, and TMPRSS2+ in villi, respectively.

Article Snippet: Tissue sections of 5 μM thickness were stained by incubating for 1h with rabbit polyclonal anti-ACE2 antibodies (Sino Biologicals, USA), then washed and stained for 40 min with Alexa Flour 568-conjugated secondary antibodies (Life Technologies, USA) ( ).

Techniques: Expressing, Infection, Two Tailed Test, Fluorescence

Journal: Frontiers in Immunology

Article Title: Simian Immunodeficiency Virus Infection Mediated Changes in Jejunum and Peripheral SARS-CoV-2 Receptor ACE2 and Associated Proteins or Genes in Rhesus Macaques

doi: 10.3389/fimmu.2022.835686

Figure Lengend Snippet: Dynamics of different renin angiotensin system proteins, lactate dehydrogenase, and cytokine production in plasma/serum. (A) Plasma concentration of ACE2 (ng/mL) at different time points of infection. The plasma ACE2 gradually decreased from acute to chronic infection with the lowest concentration at 90 dpi, then recovered during the late chronic stage of infection. (B) Plasma concentration of Angiotensin II (Ang II, pg/mL) at different time points. Though not significant, post infection showed a slight increase of Ang II compared to the pre infection time point, except at 112 and 145 dpi. (C) Plasma concentration of angiotensin II receptor 1 (AGTR1) (ng/mL) during infection. A gradual decrease of plasma AGTR1 was detected from 14 dpi onward. (D) Serum LDH activity (U/L) in infected RMs and uninfected controls were evaluated using a Beckman Coulter AU 480 analyzer. No significant changes in serum LDH activity were detected across the different time points. Plasma IL-6 (E) , IL-1β (F) , and TNF-α (G) concentrations (pg/mL) at pre and post infection time points were evaluated by U-plex biomarker NHP multiplex assay. No significant changes were detected at any time points for any of the proinflammatory cytokines tested. The error bars represent the mean ± SE for each time point (n=10). Each symbol represents individual macaque in each plot. Asterisks indicate statistical differences between time points, as calculated by Bonferroni for ACE2 and Ang II and Tukey-Kramer for AGTR1 (* p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001).

Article Snippet: Tissue sections of 5 μM thickness were stained by incubating for 1h with rabbit polyclonal anti-ACE2 antibodies (Sino Biologicals, USA), then washed and stained for 40 min with Alexa Flour 568-conjugated secondary antibodies (Life Technologies, USA) ( ).

Techniques: Concentration Assay, Infection, Activity Assay, Biomarker Assay, Multiplex Assay

Journal: Frontiers in Immunology

Article Title: Simian Immunodeficiency Virus Infection Mediated Changes in Jejunum and Peripheral SARS-CoV-2 Receptor ACE2 and Associated Proteins or Genes in Rhesus Macaques

doi: 10.3389/fimmu.2022.835686

Figure Lengend Snippet: Reduced ACE2 expression detected in lung during acute infection. Representative isotype control for ACE2 showing the absence of nonspecific background staining in lung ( A , RM FF25, 10x objective). Representative immunofluorescence images of ACE2 expression detected during pre ( B, RM FF25), acute (21 dpi in EM64, C ), chronic ( D , 180 dpi in KP54, 10x objective), and chronic ( E , 180 dpi in KP54, 20x objective) SIV infection. Expressions of ACE2 proteins in the bronchiole epithelium are shown by black arrows. (F) Scatter plots (with means ± SE) showing percentages of ACE2 positive tissue per total ROI in the bronchiole epithelium. Each point represents the average percentage of ACE2 positive tissue area from different bronchioles of each individual. ACE2 expression was reduced during acute infection, but the values were not statistically significant. ACE2 expression reverted to normal levels in chronic infection and remained higher compared to acute infection (n=6).

Article Snippet: Tissue sections of 5 μM thickness were stained by incubating for 1h with rabbit polyclonal anti-ACE2 antibodies (Sino Biologicals, USA), then washed and stained for 40 min with Alexa Flour 568-conjugated secondary antibodies (Life Technologies, USA) ( ).

Techniques: Expressing, Infection, Staining, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Simian Immunodeficiency Virus Infection Mediated Changes in Jejunum and Peripheral SARS-CoV-2 Receptor ACE2 and Associated Proteins or Genes in Rhesus Macaques

doi: 10.3389/fimmu.2022.835686

Figure Lengend Snippet: Schematic representation of the major findings. The study is stratified into three different groups: gene expression profiling of important RAS-associated proteins in Jejunum tissue during SIV infection in RM using real-time PCR; transcriptomic study of enteroids from pre and chronic SIV infected RM to identify differentially expressed gene involved in RAS; detection of RAS-associated protein expression in jejunum tissue. Gene expression analysis of jejunum tissue revealed significant differential expression of ACE2 , AGTR2 , and IL-6 gene after SIV infection. RAS-associated DEGs identified in our global transcriptomic analysis were significantly downregulated in enteroids from crypts of SIV infected RMs. In addition to the loss of CD4+ LPL, the expression of TMPRSS2 protein in jejunum villi was also downregulated. Jejunum villi AGTR2 and plasma MCP-1 protein were upregulated in SIV infection. Blue and red arrows suggest a significant downregulated and upregulated genes or proteins, respectively.

Article Snippet: Tissue sections of 5 μM thickness were stained by incubating for 1h with rabbit polyclonal anti-ACE2 antibodies (Sino Biologicals, USA), then washed and stained for 40 min with Alexa Flour 568-conjugated secondary antibodies (Life Technologies, USA) ( ).

Techniques: Expressing, Infection, Real-time Polymerase Chain Reaction

Journal: Biological Procedures Online

Article Title: Investigation of Interaction between the Spike Protein of SARS-CoV-2 and ACE2-Expressing Cells Using an In Vitro Cell Capturing System

doi: 10.1186/s12575-021-00153-9

Figure Lengend Snippet: Construction of HEK293T cells line that continuously expressing ACE2-GFP. HEK293Tcells were transfected with pCMV-ACE2-GFPSark tag plasmid and selected with hygromycin B to generate cell lines expressingACE2-GFP, cells were passaged 10 times to ensure stable expression. a Confocal imaging of HEK293T cell line with stably expressed ACE2-GFP. b Cell lysates were analyzed by Western blot with antibodies specific for ACE2 to confirm the expression of ACE2-GFP in HEK293T cells

Article Snippet: ACE2-GFP transfected HEK293T cell at 70% confluent were washed with PBS, fixed for 10 min with cold 100% methanol, and blocked using 5% BSA in PBS for 2 h. Subsequently, cells were incubated with a 1:2000 diluted anti-ACE2 rabbit polyclonal (10108-T24, Sino Biological) overnight at 4 °C, washed with PBS and incubated for 2 h with Alexa 488-conjugated goat anti-rabbit IgG antibody (Invitrogen).

Techniques: Expressing, Transfection, Plasmid Preparation, Imaging, Stable Transfection, Western Blot

Journal: Biological Procedures Online

Article Title: Investigation of Interaction between the Spike Protein of SARS-CoV-2 and ACE2-Expressing Cells Using an In Vitro Cell Capturing System

doi: 10.1186/s12575-021-00153-9

Figure Lengend Snippet: Establishment of the in vitro cell capturing system using immobilized spikeS1 protein. a - e , Optimization of the dose of immobilized spike S1 protein for capturing HEK293T/ACE2-GFP cells. Different amounts of S1 protein were coated on the 96-well microplate to capture the HEK293T/ACE2-GFP cells. Representative micrographs of captured cells are shown for 0 μg (A),0.125 μg(B),0.25 μg (C),0.5 μg(D),and 1.0 μg(E). F Quantification of captured cells using CCK8 test, data are presented as mean ± SD

Article Snippet: ACE2-GFP transfected HEK293T cell at 70% confluent were washed with PBS, fixed for 10 min with cold 100% methanol, and blocked using 5% BSA in PBS for 2 h. Subsequently, cells were incubated with a 1:2000 diluted anti-ACE2 rabbit polyclonal (10108-T24, Sino Biological) overnight at 4 °C, washed with PBS and incubated for 2 h with Alexa 488-conjugated goat anti-rabbit IgG antibody (Invitrogen).

Techniques: In Vitro

Journal: Biological Procedures Online

Article Title: Investigation of Interaction between the Spike Protein of SARS-CoV-2 and ACE2-Expressing Cells Using an In Vitro Cell Capturing System

doi: 10.1186/s12575-021-00153-9

Figure Lengend Snippet: Competitive curve of cell capturing inhibition by RBD domain of Spike protein. 0.5 μg Spike S1 protein was coated on the microplate to capture the HEK293T/ACE2-GFP cells in presence of different concentrations of spike RBD protein. Amount of captured cells were determined by CCK8 test and expressed as relative value by setting the capability to capture cells of the non-RBD group as 100%. Data were plotted with a four-parameter logistic (4PL) regression curve fit of relative cell number (y-axis) versus the competitor concentration (x-axis)

Article Snippet: ACE2-GFP transfected HEK293T cell at 70% confluent were washed with PBS, fixed for 10 min with cold 100% methanol, and blocked using 5% BSA in PBS for 2 h. Subsequently, cells were incubated with a 1:2000 diluted anti-ACE2 rabbit polyclonal (10108-T24, Sino Biological) overnight at 4 °C, washed with PBS and incubated for 2 h with Alexa 488-conjugated goat anti-rabbit IgG antibody (Invitrogen).

Techniques: Inhibition, Concentration Assay

Journal: Biological Procedures Online

Article Title: Investigation of Interaction between the Spike Protein of SARS-CoV-2 and ACE2-Expressing Cells Using an In Vitro Cell Capturing System

doi: 10.1186/s12575-021-00153-9

Figure Lengend Snippet: Correlation between expression of ACE2 on target cells and cell capturing ability. Endogenous expression of ACE2 in different cell lines determined by A Western blotting and B qPCR. C The cell capture ability of immobilized S1 to different cell lines. Captured cells were counted manually under the optical microscope; D correlation between ACE2 RNA level and S1 captured cell number fit a linear relationship with R 2 = 0.82

Article Snippet: ACE2-GFP transfected HEK293T cell at 70% confluent were washed with PBS, fixed for 10 min with cold 100% methanol, and blocked using 5% BSA in PBS for 2 h. Subsequently, cells were incubated with a 1:2000 diluted anti-ACE2 rabbit polyclonal (10108-T24, Sino Biological) overnight at 4 °C, washed with PBS and incubated for 2 h with Alexa 488-conjugated goat anti-rabbit IgG antibody (Invitrogen).

Techniques: Expressing, Western Blot, Microscopy

Journal: Biological Procedures Online

Article Title: Investigation of Interaction between the Spike Protein of SARS-CoV-2 and ACE2-Expressing Cells Using an In Vitro Cell Capturing System

doi: 10.1186/s12575-021-00153-9

Figure Lengend Snippet: The D614G mutation increases ACE2-expressing cell capturing ability of S protein. A S1 or D614G S1 variant was immobilized on 96 well microplates, and HEK293 cells stably expressing ACE2-GFP were incubated with ACE2-293 T cells. Captured cells were detached by Trypsin and counted by CCK8 test. Quantitation data are presented as mean ± SD; *** p < 0.05 by unpaired Student t-test. B FLAG-tagged ACE2 stable expressed cells lysate were incubate with Spike S1 or S1(D614G) proteins. After FLAG-IP, the immunocomplexes were blotted with anti-FLAG or anti-spike antibodies as indicated. Spike S1 monomer (apparent molecular weight ~ 116Kd) and trimer (apparent molecular weight ~ 300Kd) were found after the western blotting. Fluorescence intensity quantification of S1 or S1(D614G) indicated below each band were normalized by each input.

Article Snippet: ACE2-GFP transfected HEK293T cell at 70% confluent were washed with PBS, fixed for 10 min with cold 100% methanol, and blocked using 5% BSA in PBS for 2 h. Subsequently, cells were incubated with a 1:2000 diluted anti-ACE2 rabbit polyclonal (10108-T24, Sino Biological) overnight at 4 °C, washed with PBS and incubated for 2 h with Alexa 488-conjugated goat anti-rabbit IgG antibody (Invitrogen).

Techniques: Mutagenesis, Expressing, Variant Assay, Stable Transfection, Incubation, Quantitation Assay, Molecular Weight, Western Blot, Fluorescence

Journal: Biological Procedures Online

Article Title: Investigation of Interaction between the Spike Protein of SARS-CoV-2 and ACE2-Expressing Cells Using an In Vitro Cell Capturing System

doi: 10.1186/s12575-021-00153-9

Figure Lengend Snippet: Detachment and purification of captured cells. A A schematic overview of a 2-step cell purification protocol. In the first step, captured cells were invertedly centrifuged to remove the weakly-bound cell. In the second step, remaining cells were detached by treatment of trypsin. B Expression of ACE2 on transiently transfected ACE2-293 T cells before (left panel) and after (right panel) purification and gate M1 indicates the proportion of cells with high expression of ACE2-GFP. C Cell viability of ACE2-293 T cells before and after the purification. Cell viability was determined using Annexin-V/PI staining

Article Snippet: ACE2-GFP transfected HEK293T cell at 70% confluent were washed with PBS, fixed for 10 min with cold 100% methanol, and blocked using 5% BSA in PBS for 2 h. Subsequently, cells were incubated with a 1:2000 diluted anti-ACE2 rabbit polyclonal (10108-T24, Sino Biological) overnight at 4 °C, washed with PBS and incubated for 2 h with Alexa 488-conjugated goat anti-rabbit IgG antibody (Invitrogen).

Techniques: Purification, Expressing, Transfection, Staining

Journal: Biological Procedures Online

Article Title: Investigation of Interaction between the Spike Protein of SARS-CoV-2 and ACE2-Expressing Cells Using an In Vitro Cell Capturing System

doi: 10.1186/s12575-021-00153-9

Figure Lengend Snippet: Competitive curve of cell capturing inhibition by serum purified from Spike-protein-immunized mice. Serum from S-protein immunized non-immunized mice were titrated and pre-incubated in SARS-CoV-2 Spike S1 coated wells for 1 h before the addition of ACE2-HEK293 cells. Data were plotted with a four-parameter logistic (4PL) regression curve fit of the relative amount of captured cells determined by CCK8 test (y-axis) versus 1:2n diluted serum samples (x-axis)

Article Snippet: ACE2-GFP transfected HEK293T cell at 70% confluent were washed with PBS, fixed for 10 min with cold 100% methanol, and blocked using 5% BSA in PBS for 2 h. Subsequently, cells were incubated with a 1:2000 diluted anti-ACE2 rabbit polyclonal (10108-T24, Sino Biological) overnight at 4 °C, washed with PBS and incubated for 2 h with Alexa 488-conjugated goat anti-rabbit IgG antibody (Invitrogen).

Techniques: Inhibition, Purification, Incubation

Journal: ACS Infectious Diseases

Article Title: Human Cathelicidin Inhibits SARS-CoV-2 Infection: Killing Two Birds with One Stone

doi: 10.1021/acsinfecdis.1c00096

Figure Lengend Snippet: LL37 suppresses S1 associating with ACE2 by blocking RBD. (A) Ribbon structure of LL37 (PDB: 2K6O) in lipid micelles. (B) IC 50 determination. Results shown as the mean ± standard deviation (SD) were processed by a nonlinear curve fit. (C) Binding kinetics for LL37 and RBD. (D) Binding kinetics for ACE2 and RBD. (E) BLI-based RBD blocking assay. (F) Immunofluorescence microscopy revealing the inhibition of LL37 on S1 (Green) adhering to A549 cells and the coating of LL37 (Green) on the cell membrane. The region of interest in the S1-treated group is magnified in the embedding graph. The scale bar indicates 20 μm. (G) Protein bands of S1 pretreated with increasing concentrations of LL37 binding to A549 cells. β-actin is the reference.

Article Snippet: ACE2, the Flag tag, and the His tag were detected using an anti-ACE2 rabbit polyclonal primary antibody (10108-T24, Sino Biological, 1:1000), the anti-Flag tag rabbit monoclonal primary antibody (1:1000), and the anti-His tag mouse monoclonal primary antibody (1:1000), respectively.

Techniques: Blocking Assay, Standard Deviation, Binding Assay, Immunofluorescence, Microscopy, Inhibition, Membrane

Journal: ACS Infectious Diseases

Article Title: Human Cathelicidin Inhibits SARS-CoV-2 Infection: Killing Two Birds with One Stone

doi: 10.1021/acsinfecdis.1c00096

Figure Lengend Snippet: Complex structures of LL37 with SARS-CoV-2 RBD (A) and ACE2 (B). RBD and ACE2 (PDB: 6M0J) are shown in the ribbon structure. LL37 is shown in the red ribbon on the right. Salt bridges, hydrogen bonds, and hydrophobic interactions are shown in orange, yellow, and purple, respectively.

Article Snippet: ACE2, the Flag tag, and the His tag were detected using an anti-ACE2 rabbit polyclonal primary antibody (10108-T24, Sino Biological, 1:1000), the anti-Flag tag rabbit monoclonal primary antibody (1:1000), and the anti-His tag mouse monoclonal primary antibody (1:1000), respectively.

Techniques:

Journal: ACS Infectious Diseases

Article Title: Human Cathelicidin Inhibits SARS-CoV-2 Infection: Killing Two Birds with One Stone

doi: 10.1021/acsinfecdis.1c00096

Figure Lengend Snippet: LL37 attenuates S1 binding to cells by cloaking ACE2. (A) Binding kinetics for LL37 and ACE2. (B) BLI-based ACE2 blocking assay. (C) Protein bands of S1 binding to A549 cells pretreated with increasing concentrations of LL37. β-actin is the reference. (D) Pseudovirion neutralization assay. Results are shown as the mean ± SD. Compared with the peptide-free group, the cells pretreated with 5 and 10 μg/mL LL37 were less sensitive to SARS-CoV-2 S pseudovirion infection. ***, P < 0.001.

Article Snippet: ACE2, the Flag tag, and the His tag were detected using an anti-ACE2 rabbit polyclonal primary antibody (10108-T24, Sino Biological, 1:1000), the anti-Flag tag rabbit monoclonal primary antibody (1:1000), and the anti-His tag mouse monoclonal primary antibody (1:1000), respectively.

Techniques: Binding Assay, Blocking Assay, Neutralization, Infection

Journal: ACS Infectious Diseases

Article Title: Human Cathelicidin Inhibits SARS-CoV-2 Infection: Killing Two Birds with One Stone

doi: 10.1021/acsinfecdis.1c00096

Figure Lengend Snippet: LL37 treatment inhibits pseudovirion infection in mouse lungs. (A) Diagrammatic drawing depicting the pseudovirion-based mouse infection model. Adenovirus, Adv ; pseudovirion, Pv. (B) Protein bands of ACE2, Flag-tag, and His-tag in Lewis cells infected by the adenoviruses and pseudovirions. β-actin is the reference. In the sham group, the cells were treated with sterile PBS. (C) EGFP mRNA expression relative to β-actin. Results are shown as the mean ± SD **, P < 0.01, compared to the sham group in which mice were treated with saline solution. (D) Protein bands of Flag-tag and His-tag in mouse lungs. β-Actin is the reference. (E) Immunofluorescence microscopy revealing the inhibition of LL37 on pseudovirion infection in vivo . The scale bar is 20 μm.

Article Snippet: ACE2, the Flag tag, and the His tag were detected using an anti-ACE2 rabbit polyclonal primary antibody (10108-T24, Sino Biological, 1:1000), the anti-Flag tag rabbit monoclonal primary antibody (1:1000), and the anti-His tag mouse monoclonal primary antibody (1:1000), respectively.

Techniques: Infection, FLAG-tag, Sterility, Expressing, Saline, Immunofluorescence, Microscopy, Inhibition, In Vivo