rabbit anti furin pabs  (Proteintech)

Journal: Emerging Microbes & Infections

Article Title: A second functional furin site in the SARS-CoV-2 spike protein

doi: 10.1080/22221751.2021.2014284

Figure Lengend Snippet: Validation of furin activation sites. A–C. Infectivity analysis of SARS-CoV-2 mutants at F1, F2, and F3 sites. Normalized chemiluminescence signals (in RLUs) in 293T-ACE2-furin cells were calculated by comparing with 293T-ACE2 cells. D. Comparison of F1 and F2 single and double mutations. E. ID50 of furin inhibitor. F–G. Infectivity analysis of F1 and F2 mutations in 293T-ACE2-furin cells, with and without cathepsin inhibitor (E64D). Chemiluminescence signals (in RLUs) were normalized against WT SARS-CoV-2. H–I. Infectivity analysis of F1 and F2 mutants. Normalized chemiluminescence signals (in RLUs) in 293T-ACE2-CathepsinL/TMPRSS2 cells were calculated by comparing with 293T-ACE2 cells. A–I. Data represent the results of three replicate experiments. Values shown indicate means ± SEM. J. WT and mutated SARS-CoV-2 pseudoviruses were centrifuged in sucrose buffer, then resuspended in PBS for SDS-PAGE. Western blotting was performed with mouse anti-S2 polyclonal antibodies. VSV-M was used as an internal control. The statistical tests were comparisons of each pseudotyped mutated virus group with pseudotyped WT virus group.

Article Snippet: Anti-HA-Tag antibody (sc-7392; Santa Cruz, Dallas, TX) was used to detect TMPRSS2 and anti-c-Myc antibody (sc-40, Santa Cruz) was used to detect furin, while anti-ACE2 (10108-T60; Sino Biological Inc., Beijing, China) and anti-CTSL (10486-RP02 Sino Biological Inc.) antibodies were used to detect ACE2 and CTSL, respectively.

Techniques: Activation Assay, Infection, SDS Page, Western Blot

Journal: Emerging Microbes & Infections

Article Title: A second functional furin site in the SARS-CoV-2 spike protein

doi: 10.1080/22221751.2021.2014284

Figure Lengend Snippet: Analysis of furin activation site on cell–cell fusion. A. 293T-ACE2-furin cells were transfected with indicated S-expressing plasmid. The Cell morphology was investigated under bright field microscopy. B. Diagram of dual reporter cell–cell fusion system. 293 T cells were used as donor cells and 293T-ACE2 cells were used as recipient cells. F. Time course curve of cell–cell fusion. RLU signals of Renilla luciferase are shown. Data indicate means ± SEM. Representative results of three independent experiments are shown.

Article Snippet: Anti-HA-Tag antibody (sc-7392; Santa Cruz, Dallas, TX) was used to detect TMPRSS2 and anti-c-Myc antibody (sc-40, Santa Cruz) was used to detect furin, while anti-ACE2 (10108-T60; Sino Biological Inc., Beijing, China) and anti-CTSL (10486-RP02 Sino Biological Inc.) antibodies were used to detect ACE2 and CTSL, respectively.

Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Microscopy, Luciferase

Journal: Emerging Microbes & Infections

Article Title: A second functional furin site in the SARS-CoV-2 spike protein

doi: 10.1080/22221751.2021.2014284

Figure Lengend Snippet: Analysis of furin activation site in other coronaviruses. A. Schematic of SARS-CoV-2 S protein and the alignment of SARS-CoV-2, RaTG13, and PCoV-GD/GX at S1/S2 and S2′ sites. B–D. Infectivity of mutated RaTG13 (B), PCoV-GD (C), and PCoV-GX (D) pseudoviruses in 293T-ACE2, 293T-ACE2-furin, and 293T-ACE2-TMPRSS2 cells. RLU signals were normalized to 293T-ACE2 control cells. +F indicates that PRRA was inserted into the viruses at the S1/S2 site. The statistical tests were comparisons of each pseudotyped mutated virus group with pseudotyped WT virus group.

Article Snippet: Anti-HA-Tag antibody (sc-7392; Santa Cruz, Dallas, TX) was used to detect TMPRSS2 and anti-c-Myc antibody (sc-40, Santa Cruz) was used to detect furin, while anti-ACE2 (10108-T60; Sino Biological Inc., Beijing, China) and anti-CTSL (10486-RP02 Sino Biological Inc.) antibodies were used to detect ACE2 and CTSL, respectively.

Techniques: Activation Assay, Infection

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