Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: ( A ) Sialidase protocol validation. All lectins were directly conjugated with Alexa dyes. They were incubated with cells at 1–5 µg/mL for 15 min before a quick wash and cytometry measurement. Compared to untreated control (left), sialidase treatment (right) decreased SNA lectin binding to α2,6 sialylated structures by 15-fold and increased ECL binding to desialylated lactosamine chains (Galβ1,4GlcNAcβ) by an order of magnitude. ( B ) Pseudovirus assay. DsRed fluorescence in HEK293T and stable 293T/ACE2 cells upon addition of VSVG, Spike-WT and Spike-mutant pseudotyped virus. ( C ) Sialidase treatment of pseudovirus. % DsRed positive cell data are shown for study in (main manuscript). Viral entry was sialidase independent. ( D ) Sialidase treatment of HEK/ACE2 cells. Pseudovirus expressing VSVG, Spike-WT and Spike-mutant were added to cells under conditions described in (main manuscript). All error bars are standard deviations. Data are representative of 3 independent runs.

Article Snippet: Antibody , Anti-human F(ab’)two conjugated with Alexa Fluor FITC (goat polyclonal) , Jackson ImmunoResearch , Cat# 109-096-098 RRID: AB_2337668 , Binding assay (1:150).

Techniques: Biomarker Discovery, Incubation, Cytometry, Control, Binding Assay, Fluorescence, Mutagenesis, Virus, Expressing

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: A panel of lectins (from Vector Labs) was conjugated with Alexa dyes, either Alexa 405, 488 or 647. The binding of these fluorescent reagents to wild-type 293T, [N] - 293T and [O] - 293 T cells was measured using flow cytometry. The lectins bound: ( A ) N-glycan high-mannose and complex structures [ConA and LCA bind αMan in high-mannose glycans; PHA-L and PHA-E bind complex glycans], ( B ) lactosamine chains primarily on N-linked glycans [RCA, ECL bind terminal Gal or lactose; DSL bind β1,4GlcNAc], and ( C ) O-glycan related structures [PNA binds Galβ1,GalNAc; VVA and SBA bind GalNAcα]. Measurements were made with either untreated or sialidase treated 293 T cells. As seen: i. Knocking out MGAT1 in [N] - 293T reduces lectin binding in panels A and B (see arrow). ii. Knocking out C1GalT1 results in a dramatic decrease in PNA binding and increase in VVA and SBA binding, These data are consistent with the expected changes in lectin profile upon knocking out these N- and O-glycan-specific enzymes.

Article Snippet: Antibody , Anti-human F(ab’)two conjugated with Alexa Fluor FITC (goat polyclonal) , Jackson ImmunoResearch , Cat# 109-096-098 RRID: AB_2337668 , Binding assay (1:150).

Techniques: Plasmid Preparation, Binding Assay, Flow Cytometry, Glycoproteomics

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-human F(ab’)two conjugated with Alexa Fluor FITC (goat polyclonal) , Jackson ImmunoResearch , Cat# 109-096-098 RRID: AB_2337668 , Binding assay (1:150).

Techniques: Binding Assay, Plasmid Preparation, Recombinant, Knock-Out, Derivative Assay

Journal: Cancer biology & therapy

Article Title: Cancer gene therapy of adenovirus-mediated anti-4-1BB scFv in immunocompetent mice.

doi: 10.4161/cbt.7.3.5425

Figure Lengend Snippet: Figure 2. Characterization of anti-4-1BB scFv expression. (A) RT-PCR analy- sis of anti-4-1BB scFv mRNA expression. Cultured cells were infected with Ad.CMV.AP or Ad.4-1BB scFv at a MOI of 10 for 24 h. The transcripts of anti- 4-1BB scFv were semi-quantified by RT-PCR with β-actin as an internal control. (B) Flow cytometric analysis of anti-4-1BB scFv cell surface expression. Hepa 1-6 cells were infected with Ad.4-1BB scFv or Ad.CMV.AP at a MOI of 10 for 24 h and stained with FITC-conjugated anti-human IgG F(c) antibody that recognized the immunoglobulin Fc fragments expressed on Ad.4-1BB scFv infected Hepa1-6 cells for flow cytometry analysis. (C) Visualizing anti-4-1BB scFv expression on the surface of Hepa1-6 cells by direct immunofluorescence staining. Cells were infected with Ad.4-1BB scFv or Ad.CMV.AP at a MOI of 30 for 24 h, fixed, incubated with FITC-conjugated anti-human IgG F(c) anti- body, and counterstained with Hoechst 33342. The same optical field was photographed under fluorescence light and normal light. The blue fluores- cence corresponded to cell nuclei, and the green fluorescence corresponded to anti-4-1BB scFv-hIgG Fc. Original magnification, x630.

Article Snippet: After 24 h of infection, cells were harvested, washed twice with ice-cold PBS and incubated in a 1:250 dilution of a fluorescein isothiocyanate (FITC)-labeled goat anti-human IgG F(c) (Rockland Immunochemicals Inc., PA, USA) on ice for 1 h, which recognized the immunoglobulin Fc fragments expressed on virus infected Hepa1-6 cells.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Infection, Control, Staining, Flow Cytometry, Immunofluorescence, Incubation, Fluorescence

Journal: Cell

Article Title: Anti-NKG2A mAb Is a Checkpoint Inhibitor that Promotes Anti-tumor Immunity by Unleashing Both T and NK Cells

doi: 10.1016/j.cell.2018.10.014

Figure Lengend Snippet: NKG2A Is an Inhibitory Receptor that Blocks the Anti-tumor Efficacy of NK and CD8 + T Cells (A) Qa-1 b -sufficient or -deficient A20 tumor cells were engrafted subcutaneously (s.c.) in BALB/c mice. (B) BALB/c mice were treated with an anti-aGM1 pAbs or with control rabbit serum, an anti-CD8α mAb, or rat IgG2b isotype control and then subcutaneously engrafted with A20 tumor cells. Graphs show tumor growth in each individual mouse and combined survival curves. Complete regressions are indicated. log rank test, ∗∗ p = 0.0020; ns, no significant. (C) Experiment similar to that in (B), but with Qa-1 b KO A20 tumor cells. Complete regressions are indicated. log rank test, ∗∗∗ p = 0.0002 (NK cell depletion) and ∗∗∗ p = 0.0006 (CD8 + T cell depletion). See also Figure S1 .

Article Snippet: Peripheral blood cells were then stained with anti-CD3-PercpCy5.5 (BD Biosciences), anti-CD45-APC (BD Biosciences), anti-CD3-PC7 (BD Biosciences) and anti-human IgG4 Fc-FITC (HP6025, Southern Biotechnology).

Techniques: Control

Journal: Cell

Article Title: Anti-NKG2A mAb Is a Checkpoint Inhibitor that Promotes Anti-tumor Immunity by Unleashing Both T and NK Cells

doi: 10.1016/j.cell.2018.10.014

Figure Lengend Snippet:

Article Snippet: Peripheral blood cells were then stained with anti-CD3-PercpCy5.5 (BD Biosciences), anti-CD45-APC (BD Biosciences), anti-CD3-PC7 (BD Biosciences) and anti-human IgG4 Fc-FITC (HP6025, Southern Biotechnology).

Techniques: Purification, Control, Recombinant, Selection, Staining, Software