furin antibodies  (R&D Systems)

Journal: PLOS Pathogens

Article Title: Antibody-mediated spike activation promotes cell-cell transmission of SARS-CoV-2

doi: 10.1371/journal.ppat.1011789

Figure Lengend Snippet: ( A ) Immunoblots showing shedded S1 subunits and human IgG heavy chain (IgG Hc), full-length spike, S1, S2 and cleaved S2’ collected from supernatant and cell lysate fractions of antibody-treated HEK293T cells expressing WT full-length spike for 16 hours. CB6, FD20, REGN10933 and REGN10987 were diluted in PBS and used at 12.5 nM, blots are representative of at least three independent experiments; ( B ) Immunoblots showing proteinase K-resistant S2’ cleavage product, obtained from cell lysates described in (A) and were then treated in the absence or presence of 10 μg/mL proteinase K for 30 min at 37°C, blots are representative of two individual repeats; ( C ) Luciferase activity (RLU) measured from cell-cell fusion assay and immunoblots showing shedded S1 subunits, IgG Hc, full-length spike, S1, S2 and cleaved S2’ collected from supernatant and cell lysate fractions of CB6-stimulated HEK293T cells. CB6 doses used were 0.1, 0.5, 2.5 and 12.5 nM respectively, human IgG isotype was used at 12.5 nM for the negative control. Data and blots are representative of four individual repeats; ( D ) Immunoblots showing shedded S1 subunits, full-length spike and S1 collected from supernatant and cell lysate fractions of lentivirus-transduced A549 cells expressing WT full-length spike, stimulated without or with 12.5 nM CB6 antibody. Blots are representative of three individual repeats; ( E ) Luciferase activity (RLU) measured from cell-cell fusion assay and immunoblots showing shedded S1 subunits, IgG Hc, full-length spike, S1, S2 and cleaved S2’ collected from supernatant and cell lysate fractions of CB6-stimulated HEK293T cells expressing Beta spike VOCs carrying N417 or K417 revert mutation, blots and data are representative of four individual repeats; ( F ) Schematics of the R685A (furin-cleavage site deficient) spike mutant, and representative fluorescent images captured at 594 nm and 405 nm from HEK293T cells expressing WT and R685A spike mutant, stimulated without or with 12.5 nM CB6; scale bars are representative of 50 μm, images were representative of two individual repeats; ( G ) Luciferase activity (RLU) measured from antibody-induced cell-cell fusion assay, where co-cultured HEK293T cells expressing WT or R685A spike mutant were stimulated without or with 12.5 nM CB6 for 16 hours (Top). Supernatants and cell lysates were used for immunoblots of shedded S1 subunits, IgG Hc, full-length spike, S1, S2 and cleaved S2’ (Bottom). Blots are representative of three independent experiments.

Article Snippet: Rabbit anti-PARP antibody (9532S), anti-EEA1(3288S) and rabbit anti-LAMP1 (9091S) were purchased from CST, and rabbit anti-human furin polyclonal antibody (18413-1-AP) was purchased from Proteintech.

Techniques: Western Blot, Expressing, Luciferase, Activity Assay, Cell-Cell Fusion Assay, Negative Control, Mutagenesis, Cell Culture

Journal: PLOS Pathogens

Article Title: Antibody-mediated spike activation promotes cell-cell transmission of SARS-CoV-2

doi: 10.1371/journal.ppat.1011789

Figure Lengend Snippet: ( A ) Immunoblots of shedded S1 subunits, IgG Hc collected from supernatants; or full-length spike, S1, S2 and cleaved S2’ and IgG Hc collected from HEK293T cell lysates expressing WT or R685A spike mutant, stimulated without or with 12.5 nM CB6 antibody for 16 hours, in the absence or presence of 50 nM Bafilomycin A1 (BafA1). Blots are representative of three individual experiments; ( B ) Representative confocal images of 12.5 nM CB6 antibody-stimulated HEK293T cells expressing WT or R685A spike mutant for 16 hours. Anti-Early endosomes antigen 1 (EEA1) and Anti-human IgG (H+L chains) were stained with Alexa fluor 488 and 555 respectively, co-localizations are indicated with white arrows and R values derived from Pearson’s coefficients summarized, scale bars are representative of 10 μm. Images are representative of four individual experiments; ( C ) Luciferase activity (RLU) measured from 12.5 nM CB6-stimulated siControl or siFURIN donor HEK293T cells co-expressing Cre and WT spike, mixed with siControl or siFURIN acceptor Stop-Luc- expressing cells for 16 hours (top); and immunoblots showing shedded S1 subunits, hIgG Hc and full-length spike, S1, S2 and S2’ collected from co-cultured cell supernatants and lysates (bottom). Data shown are representative of four independent repeats; ( D ) Luciferase activity (RLU) measured from 12.5 nM CB6-stimulated over-expressing pcDNA4 (empty vector) or FURIN donor HEK293T cells co-expressing Cre and WT spike, mixed with over-expressing pcDNA4 or FURIN acceptor Stop-Luc -expressing cells for 16 hours (top); and immunoblots showing shedded S1 subunits, hIgG Hc and full-length spike, S1, S2 and S2’ collected from co-cultured cell supernatants and lysates (bottom). Data shown are representative of four independent repeats; ( E ) Luciferase activity (RLU) measured from HEK293T cells co-expressing WT spike and Cre, mixed with Stop-Luc -expressing cells, in the absence or presence of 25 μM dec-RVKR-cmk (RVKR) for 16 hours (top); immunoblots showing shedded S1 subunits, full-length S, S1, S2 and cleaved S2’ collected from supernatants and lysates (bottom). Data shown are representative of five independent repeats, and the blot is representative of three repeats; ( F ) Representative confocal images of 12.5 nM CB6 antibody-stimulated HEK293T cells expressing WT spike, treated with DMSO or 25 μM RVKR for 16 hours. Anti-Early endosomes antigen 1 (EEA1) and Anti-human IgG (H+L chains) were stained with Alexa fluor 488 and 555 respectively, scale bars are representative of 10 μm, co-localizations are indicated with white arrows and R values derived from Pearson’s coefficients were summarized. Data are displayed as individual points with mean ± standard error of the mean (SEM). P value was obtained by one-way ANOVA with Sidak’s post hoc test and is indicated on the figure.

Article Snippet: Rabbit anti-PARP antibody (9532S), anti-EEA1(3288S) and rabbit anti-LAMP1 (9091S) were purchased from CST, and rabbit anti-human furin polyclonal antibody (18413-1-AP) was purchased from Proteintech.

Techniques: Western Blot, Expressing, Mutagenesis, Staining, Derivative Assay, Luciferase, Activity Assay, Cell Culture, Plasmid Preparation

Journal: PLOS Pathogens

Article Title: Antibody-mediated spike activation promotes cell-cell transmission of SARS-CoV-2

doi: 10.1371/journal.ppat.1011789

Figure Lengend Snippet: ( A ) When SARS-CoV-2 spike is cleaved at the S1/S2 cleavage site and expressed on the infected cell membrane, binding of Class I antibodies (Abs) onto the receptor binding motif (RBM) trigger the rapid shedding of S1 subunit at the cell surface. This event allows the functional activation of the S2’ cleavage site by membrane bound proteases, such as TMPRSS2. Exposure of fusion peptide at the plasma membrane triggers receptor-independent cell-cell fusion among adjacent cells. This process drives the functional activation of spike-expressed on the cell membrane and promote cell-cell transmission of the SARS-CoV-2 virus; ( B ) However, when spike S1/S2 site is not cleaved by an endogenously expressed protease, for instance by host cell furin or TMPRSS2, binding of Class I Abs on the RBM is unable to trigger S1 shedding from the spike-expressing cells. Instead, binding of Class I Ab leads to the internalization of spike trimers, where S2’ cleavage and exposure of fusion peptide could occur inside endolysosomes. As a result, cell-cell transmission of the SARS-CoV-2 virus could be efficiently prevented. Figure was illustrated using images created with BioRender.com .

Article Snippet: Rabbit anti-PARP antibody (9532S), anti-EEA1(3288S) and rabbit anti-LAMP1 (9091S) were purchased from CST, and rabbit anti-human furin polyclonal antibody (18413-1-AP) was purchased from Proteintech.

Techniques: Infection, Membrane, Binding Assay, Functional Assay, Activation Assay, Clinical Proteomics, Transmission Assay, Virus, Expressing

Journal: JCI insight

Article Title: Protease-dependent defects in N-cadherin processing drive PMM2-CDG pathogenesis.

doi: 10.1172/jci.insight.153474

Figure Lengend Snippet: Figure 4. Proprotein convertase and Mmp activity is reduced in pmm2m/m embryos. (A) Schematic of protease-mediated N-cadherin cleavage. (B) In vitro enzyme assays for PCs in embryo lysates show increased activity in pmm2m/m embryos 7 dpf. n = 3 experiments of 15 embryos per condition per sample. Error bars show SEM, Dunnett’s test, **P < 0.01, ***P < 0.001. (C) Western blot of Furin enzyme in embryo lysates; P (green arrow), pro form; M (red arrow), mature form. (D) Gelatin zymography of embryos shows decrease in gelatinase activity in pmm2m/m embryos 7 dpf (red arrows). (E) Graphs quanti- tate gelatinase activity. n = 4 experiments of 15 embryos per condition per sample. Error bars show SEM, Student’s t test, ***P < 0.001. (F) Two Western blots of Mmp2. Immunoblot 2 (IB2) is shown at higher exposure with a higher magnification “inset” that illustrates the pro and mature bands. Red stars highlight a shift in Mmp2’s molecular weight in pmm2m/m embryos relative to control embryos. n = 3 experiments with 15 embryos per sample per experi- ment. (G) Two Western blots of Mmp9. In immunoblot 2 stars denote the pro (green star) and mature (red star) forms of Mmp9 present in pmm2 control embryos, while only the pro form is noted in pmm2m/m embryos. n = 3 experiments with 15 embryos per sample per experiment.

Article Snippet: Blots were probed using an N-cadherin antibody (1:1000, catalog ab211126; Abcam), an MMP2 antibody (1:750, catalog AF902; R&D Systems, Bio-Techne), an MMP9 antibody (1:500, catalog AS-55345; AnaSpec), or a Furin antibody (1:2500, catalog AF1503; R&D Systems, Bio-Techne).

Techniques: Activity Assay, In Vitro, Western Blot, Zymography, Molecular Weight, Control

Journal: Journal of Clinical Laboratory Analysis

Article Title: Increased FURIN expression in rheumatoid arthritis patients and its anti‐inflammatory effect

doi: 10.1002/jcla.23530

Figure Lengend Snippet: The primer sequence

Article Snippet: Anti‐human FURIN antibody was diluted at 1:500 (sc‐133142, Santa Cruz Biotechnology), anti‐GAPDH (#5174, Cell Signaling Technology), anti‐caspase‐1 (#2225, Cell Signaling Technology), and anti‐IL1β (#12242, Cell Signaling Technology) were diluted to a concentration of 1:1000.

Techniques:

Journal: Journal of Clinical Laboratory Analysis

Article Title: Increased FURIN expression in rheumatoid arthritis patients and its anti‐inflammatory effect

doi: 10.1002/jcla.23530

Figure Lengend Snippet: FURIN levels in patients with rheumatoid arthritis and in healthy control participants. A, Relative FURIN mRNA expression in peripheral blood mononuclear cells (PBMCs) from RA and healthy control participants, normalized against the expression of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). B, Levels of serum FURIN in patients with RA compared with those of healthy control participants. C, Relative FURIN mRNA expression in PBMCs among four RA groups, normalized against the expression of GAPDH. D, Serum FURIN levels among the four RA groups. Horizontal lines represent the median values in each group. The Mann‐Whitney U test was used for statistical comparisons. The differences among the four RA groups were assessed by using the Kruskal‐Wallis H nonparametric test. Significant differences are marked with asterisks: * P < .05, ** P < .01, and *** P < .001

Article Snippet: Anti‐human FURIN antibody was diluted at 1:500 (sc‐133142, Santa Cruz Biotechnology), anti‐GAPDH (#5174, Cell Signaling Technology), anti‐caspase‐1 (#2225, Cell Signaling Technology), and anti‐IL1β (#12242, Cell Signaling Technology) were diluted to a concentration of 1:1000.

Techniques: Control, Expressing, MANN-WHITNEY

Journal: Journal of Clinical Laboratory Analysis

Article Title: Increased FURIN expression in rheumatoid arthritis patients and its anti‐inflammatory effect

doi: 10.1002/jcla.23530

Figure Lengend Snippet: Correlation between FURIN and disease activity and clinical data

Article Snippet: Anti‐human FURIN antibody was diluted at 1:500 (sc‐133142, Santa Cruz Biotechnology), anti‐GAPDH (#5174, Cell Signaling Technology), anti‐caspase‐1 (#2225, Cell Signaling Technology), and anti‐IL1β (#12242, Cell Signaling Technology) were diluted to a concentration of 1:1000.

Techniques: Activity Assay

Journal: Journal of Clinical Laboratory Analysis

Article Title: Increased FURIN expression in rheumatoid arthritis patients and its anti‐inflammatory effect

doi: 10.1002/jcla.23530

Figure Lengend Snippet: Correlation of FURIN with transforming growth factor (TGF)‐β1. A, Correlation between FURIN mRNA levels and TGF‐β1 mRNA levels, normalized to the expression levels of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). B, Correlation of protein levels between FURIN and TGF‐β1 in serum

Article Snippet: Anti‐human FURIN antibody was diluted at 1:500 (sc‐133142, Santa Cruz Biotechnology), anti‐GAPDH (#5174, Cell Signaling Technology), anti‐caspase‐1 (#2225, Cell Signaling Technology), and anti‐IL1β (#12242, Cell Signaling Technology) were diluted to a concentration of 1:1000.

Techniques: Expressing

Journal: Journal of Clinical Laboratory Analysis

Article Title: Increased FURIN expression in rheumatoid arthritis patients and its anti‐inflammatory effect

doi: 10.1002/jcla.23530

Figure Lengend Snippet: FURIN inhibition in THP‐1‐derived macrophages upregulated IL‐1β and caspase‐1. A, Western blot of THP‐1‐derived macrophages transfected with either FURIN siRNA (si‐FURIN) or control siRNA (NC) for 24 h. B, FURIN protein levels. C, FURIN mRNA levels. D, TGF‐β1 mRNA levels. E, IL‐1β mRNA levels. F, TNF‐α mRNA levels. G, Western blot of THP‐1‐derived macrophages transfected with either FURIN siRNA (si‐FURIN) or control siRNA (NC) for 24 h, followed by incubation with LPS (1 μg/mL) for 4 h and ATP (5 mmol/L) for 30 min. (H) Caspase‐1 protein levels; (I) IL‐1β protein levels. * P < .05, ** P < .01

Article Snippet: Anti‐human FURIN antibody was diluted at 1:500 (sc‐133142, Santa Cruz Biotechnology), anti‐GAPDH (#5174, Cell Signaling Technology), anti‐caspase‐1 (#2225, Cell Signaling Technology), and anti‐IL1β (#12242, Cell Signaling Technology) were diluted to a concentration of 1:1000.

Techniques: Inhibition, Derivative Assay, Western Blot, Transfection, Control, Incubation