mab3870 r d systems 2 anti furin  (R&D Systems)

Journal: Science Advances

Article Title: Inhibition of placental trophoblast fusion by guanylate-binding protein 5

doi: 10.1126/sciadv.adt5388

Figure Lengend Snippet: ( A ) Interaction of furin or PCSK7 with GBPs. HEK293T cells were cotransfected with expression plasmids for the indicated PCSKs and GBPs or the respective vector controls. One day posttransfection, cells were harvested, and furin or PCSK7 were pulled down via their C-terminal AU1 tag. Western blotting was performed to detect PCSKs and GBPs in whole cell lysates (left) and the pull down (right). The results of one of three independent experiments are shown. IP, immunoprecipitation; #, protein size marker. ( B and C ) Furin- and PCSK7-mediated Syncytin cleavage in the presence of GBPs. Furin and PCSK7 were captured from cell lysates of transfected HEK293T cells essentially as described in . However, cells were additionally cotransfected with expression plasmids for GBP2 or GBP5 (WT or isoprenlyation mutant) or the respective vector control. The ability of captured furin/PCSK7 to process a consensus furin cleavage site (B) or the Syncytin-1 and Syncytin-2 cleavage sites (C) were analyzed using the AMC reporter assay described in . Mean values of three independent experiments ± SD are shown on top. A one-way ANOVA with multiple comparisons (Dunnett’s test) was performed (* P < 0.05, ** P < 0.01, and *** P < 0.001). Capture efficiency of PCSKs was monitored by Western blotting and is shown at the bottom.

Article Snippet: The membranes were stained using primary antibodies directed against HA tag (Abcam, catalog no. ab18181), AU1-tag (Novus Biologicals, catalog no. NB600-453), V5-tag (Cell Signaling, catalog no. 13202), GBP5 (Santa Cruz, catalog no. sc-160353), furin (R&D Systems, catalog no. AF1503), GAPDH (BioLegend, catalog no. 607902), and infrared dye-labeled secondary antibodies (LI-COR IRDye).

Techniques: Expressing, Plasmid Preparation, Western Blot, Immunoprecipitation, Marker, Transfection, Mutagenesis, Control, Reporter Assay

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